Methods and compositions for treating mammalian nerve tissue injuries

ABSTRACT

To achieve, an in vivo repair of injured mammalian nerve tissue, an effective amount of a biomembrane fusion agent is administered to the injured nerve tissue. The application of the biomembrane fusion agent may be performed by directly contacting the agent with the nerve tissue at the site of the injury. Alternatively, the biomembrane fusion agent is delivered to the site of the injury through the blood supply after administration of the biomembrane fusion agent to the patient. The administration is preferably by parenteral administration including including intravascular, intramuscular, subcutaneous, or intraperitoneal injection of an effective quantity of the biomembrane fusion agent so that an effective amount is delivered to the site of the nerve tissue injury.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/132,542, filed Apr. 24, 2002, which claims the benefit ofU.S. Provisional Patent Application No. 60/286,200 filed Apr. 24, 2001,both of which are hereby incorporated by reference in their entirety.

This invention was made in part with government support under grantnumber DAMDI 7-94-J-4242 awarded by the Department of the Army and grantnumber BES9631560 awarded by the National Science Foundation. Furtherfinancial support for development of this invention was provided by NSFgrant CCR 92-22467 and NIH grant ROI NS39288-01. The Government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to methods for treating injuredmammalian nerve tissue including but not limited to a spinal cord.Specifically, the invention relates to methods for treating injurednerve tissue through an in vivo application of a biomembrane fusionagent. Pharmaceutical compositions for treating an injured spinal cordare also described.

BACKGROUND OF THE INVENTION

Mechanical damage to the nervous system of mammals results in sometimesirreversible functional deficits. Most functional deficits associatedwith trauma to both the Peripheral Nervous System (PNS) or CentralNervous System (CNS) result from damage to the nerve fiber or axon,blocking the flow of nerve impulse traffic along the nerve fiber. Thismay be due to a physical discontinuity in the cable produced by axotomy.The blockage may also occur where the membrane no longer functions as anionic fence, and/or becomes focally demyelinated [Honmou, O. and Young,W. (1995) Traumatic injury to the spinal axons (Waxman, S. G., Kocsis,J. D., Stys, P. K., Eds.): The Axon, New York: Oxford UP, pp 480-503;Maxwell, W. L. (1996): Histopathological changes at central nodes ofravier after stretch-injury, Microscopy Research and Technique, 34:522-535; Maxwell, W. L., Watt, C., Graham, D. I., Gennarelli, T. A.(1993): Ultrastructural evidence of axonal shearing as a result oflateral acceleration of the head in non-human primates, ActaNeuropathol, 86: 136-144; Maxwell, W. L., Graham, D. I. (1997): Loss ofaxonal microtubules and neurofilaments after stretch-injury to guineapig optic nerve fibers, J Neurotrauma, 14: 603-614; Blight, A. R.(1993): Remyelination, Revascularization, and Recovery of Function inExperimental Spinal Cord Injury (Seil, F. J., Ed.): Advances inNeurobiology: Neural Injury and Regeneration, Vol. 59, New York, RavenPress, pp. 91-103]. In either case, functional deficits occur because ofthe break in nerve impulse conduction. Even the severe behavioraldeficits associated with spinal cord injury is now understood to belargely due to the initial mechanical damage to white matter [Blight, A.R.: Morphometric analysis of a model of spinal cord injury in guineapigs, with behavioral evidence of delayed secondary pathology, J.Neurolog. Sci., 103: 156-171, 1991]. Delayed but progressive episodes ofso-called “secondary injury” [Honmou and Young, W. (1995): Traumaticinjury to the spinal axons (Waxman, S. G., Kocsis, J. D., Stys, P. K.,Eds.): The Axon, New York: Oxford UP pp 480-503; Young, W. (1993):Secondary injury mechanisms in acute spinal cord injury, J. Emerg. Med.,11: 13-22.] subsequently enlarge the lesion leading to the typicalclinical picture of a cavitated contused spinal cord, and intractablebehavioral loss.

In the mammal, transection of the axon leads to the irreversible loss ofthe distal nerve process segment by Wallerian degeneration, while theproximal segment may survive. In the PNS, function may be restored bythe endogenous regeneration of proximal segments down fasciculationpathways provided by both connective tissue and Schwann cell “tubes”which may persist for variable amounts of time post injury (Bisby, M. A.(1995): Regeneration of peripheral nervous system axons (Waxman, S. G.,Kocsis, J. D., Stys, P. K., Eds.): The Axon Book, New York, The OxfordUniversity Press, pp 553-578]. The level of the injury is critical toclinical fascicular repair however, as the rate of regeneration (about 1mm/day) may not be sufficient to avoid loss of target tissues dependenton its innervation (such as motor units in striated muscle). In the CNS,distal segments of nerve fibers do not regenerate, and their lossproduces nonfunctional “target” cells, which often require innervationto maintain their integrity. One ultimate strategy to enhance recoveryfrom CNS injury is to induce or facilitate regeneration of white matterby various means.

In the clinic, acute spinal cord transection is rare whilecompressive/contusive mechanical damage is typical. In the PNS,transection, stretch injury as well as compression injury to nervetrunks are commonplace. However, severe, local, mechanical damage to anytype of nerve fiber membrane may still initiate a process leading toaxotomy and the irretrievable loss of distal segments. These eventsusually begin with a breakdown in the ability of the axolemma toseparate and maintain critical differences in ions between theextracellular and intracellular compartments—in particular calcium.

The devastating effects of injury to the mammalian spinal cord are notimmediate. Severe mechanical injury initiates a delayed destruction ofspinal cord tissue producing a loss in nerve impulse conductionassociated with a progressive local dissolution of nerve fibers (axons)[Honmou, O. and Young, W. (1995) The Axon (Waxman, S. G., et al., Eds.)pp. 480-529, Oxford University Press, New York; Griffin, J. W. et al.(1995) The Axon (Waxman, S. G., et al., Eds.) pp. 375-390, OxfordUniversity Press, New York]. This loss of sensory and motorcommunication across the injury site can produce a permanent paralysisand loss of sensation in regions below the level of the spinal injury.Furthermore, it is clear the most damaging effects of progressive“secondary injury” [Young, W. (1993) J. Emerg. Med. 11: 13-22] of spinalcord parenchyma relative to the loss of behavioral functioning is theeffect it has on white matter. Localized mechanical, biochemical, andanoxic/ischemic injury to white matter may be sufficient to cause thefailure of axolemmas to function as a barrier or fence to theunregulated exchange of ions [Honmou, O. and Young, W. (1995) The Axon(Waxman, S. G., et al., Eds.) pp. 480-529, Oxford University Press, NewYork]. This in turn compromises both the structural integrity of thisregion of the nerve fiber and its ability to conduct impulses along thecable. For example, elevated intracellular Ca²⁺ induces depolymerizationof microtubules and microfilaments producing a focal destruction of thecytoskeleton [Griffin, J. W. et al. (1995) The Axon (Waxman, S. G., etal., Eds.) pp. 375-390, Oxford University Press, New York; Maxwell, W.L., et al. (1995) J. Neurocytology 24: 925-942; Maxwell, W. L., et al.J. Neurotrauma 16: 273-284].

The unrestricted movement of Ca⁺⁺ down its electrochemical gradient intothe cell leads to a destruction of membranes and the cytosol, and is aninitial key event in all mechanical injury to nerve fibers as well asother ischemic injuries such as head injury and stroke [Borgens, R. B.,Jaffe, L. F., Cohen, M. J. (1980): Large and persistent electricalcurrents enter the transected spinal cord of the lamprey eel, Proc.Natl. Acad. Sci. U.S.A., 77: 1209-1213; Borgens, R. B. (1988): Voltagegradients and ionic currents in injured and regenerating axons, Advancesin Neurology, 47: 51-66; Maxwell, W. L. (1996): Histopathologicalchanges at central nodes of ravier after stretch-injury, MicroscopyResearch and Technique, 34: 522-535; Maxwell, W. L., Graham, D. I.(1997): Loss of axonal microtubules and neurofilaments afterstretch-injury to guinea pig optic nerve fibers, J. Neurotrauma, 14:603-614; Maxwell, W. L., Watt, C., Graham, D. I., Gennarelli, T. A.(1993): Ultrastructural evidence of axonal shearing as a result oflateral acceleration of the head in non-human primates, ActaNeuropathol, 86: 136-144; Honmou and Young, 1995, Lee et al., 1999; Styset. al., 1990]. Na⁺ enters the localized region of the membrane insultas well, depolarizing the membrane and facilitating the release ofintracellular Ca⁺⁺ stores [Carafoli, E., Crompton, M. (1976): Calciumions and mitochondria (Duncan, C. J., Ed.): Symposium of the Society forExperimental Biology: Calcium and Biological Systems, Vol. 30, New York,Cambridge University Press, pp. 89-115; Borgens, R. B., Jaffe, L. F.,Cohen, M. J. (1980): Large and persistent electrical currents enter thetransected spinal cord of the lamprey eel, Proc. Natl. Acad. Sci.U.S.A., 77: 1209-1213; 1988; Borgens, R. B. (1988): Voltage gradientsand ionic currents in injured and regenerating axons, Advances inNeurology, 47: 51-66]. Potassium exodus also pushes the restingpotential of the membrane towards the Nernst potential for K⁺contributing to the localized region of inexcitability and blockage ofnerve impulse conduction down the cable in even intact membranes. Thus,when K⁺ rushes down its electrochemical gradient out of the cell, theresultant elevated extracellular concentration contributes to localizedconduction block [Honmou, O. and Young, W. (1995) The Axon (Waxman, S.G., et al., Eds.) pp. 480-529, Oxford University Press, New York; Shi,R. et al., (1997) Society for Neuroscience Abstracts, 108: 16]. Howeverit is the progressive chain reaction of events set in motion by Ca⁺⁺entry into the cell that initially leads to progressive dissolution ofthe axon—aided in later stages of the acute event by additional complexmolecular processes such as the initiation of lipid peroxidationpathways and formation of “free radical” oxygen metabolites.

There are several classes of molecules that have already been shown tobe able to seal cell membranes or to actually fuse membranes together[Nakajima, N., Ikada, Y. (1994): Fusogenic activity of variouswater-soluble polymers, J. Biomaterials Sci., Polymer Ed., 6: 751-9].These biocompatible polymers can also resolve discontinuities in theplane of the membrane into an unbroken plasmalemma, and/or becomeinserted into the membrane defect, sealing it and reversingpermeabilization.

For over thirty years polyethylene glycol (PEG) has been known to fusemany cells together to form one giant cell. Application of thishydrophilic macromolecule has been exploited to form multicellularconjugates for the purpose of exchanging genetic material, hybridomaformation, or as a model for endogeneous vesicle fusion [Davidson, R.L., O'Malley, K. A., Wheeler, T. B. (1976): Induction of mammaliansomatic cell hybridization by polyethylene glycol, Somat. Cell Genet.,2: 271-280; Lee, J., Lentz, B. R. (1997): Evolution of lipid structuresduring model membrane fusion and the relation of this process to cellmembrane fusion, Biochemistry, 36: 6251-6259; Lentz, B. R. (1994):Induced membrane fusion; Potential mechanism and relation to cell fusionevents, Chem. and Phys. of Lipids, 73: 91-106]. PEG has also been usedto fuse many phaetocychroma cells (PC-12; neuron like cells) together toproduce large single units facilitating neurophysiological measurementsin vitro as well as fusing the severed ends of single invertebrate giantaxons in vitro [O'Lague, P. H., Huttner, S. L. (1980): Physiological andmorphological studies of rat phechromocytoma calls (PC12) chemicallyfused and grown in culture, Proc. Nat. Acad. Sci. USA, 77: 1701-1705;Krause, T. L., Bittner, G. D. (1990, 1991): Rapid morphological fusionof severed myelinated axons by polyethylene glycol, PNAS, 87:1471-1475].

Methods and compositions for treating mammalian spinal cord injuries areneeded. The present invention addresses these needs.

SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions for the invivo repair of injured mammalian nerve tissue. The invention is moreparticularly directed to the application or administration of aneffective amount of a biomembrane fusion agent (see Definitions sectionbelow) to the site of an injury (see Definitions section below) to nervetissue, particularly nerve tissue of the spinal cord or the peripheralnervous system. The biomembrane fusion agent may be directly contactedwith the nerve tissue at the site of the injury or may be administeredto the patient parenterally. Preferably, the biomembrane fusion agent isdelivered to the site of the injury through the blood supply afterinjection of the biomembrane fusion agent into the patient. Theinjection may be an intravascular, intramuscular, subcutaneous, orintraperitoneal injection of an effective quantity of the biomembranefusion agent so that an effective amount is delivered to the site of thenerve tissue injury.

Preferably, the biomembrane fusion agent takes the form of a hydrophilicpolymer in the form of a polyalkylene glycol or oxide such as apolyethylene glycol, a polyethylene glycol/polypropylene glycol blockcopolymer such as ethylene oxide-propylene oxide-ethylene oxide (EPAN),or another hydrophilic biocompatible surfactant such as dextrans. Thesurfactant is preferably nonionic and may take the form of anamphipathic polymer such as a poloxamine. Most preferably, thebiomembrane fusion agent is polyethylene glycol (PEG)(H(OCH₂CH₂)_(n)OH), where n preferably ranges from 4 to about 570 ormore, more preferably about 30 to about 100. PEG is used as a solventfor many compounds used in medicine. For example, PEG is used as acarrier for contrast media used in radiology, and a solvent forhemopoetic factors infused into hemophilic patients. A suitablealternative is a poloxamer (see Definitions section below). Some ofthese triblock polymers consist of PEG polymers with a propylene glycolcore. The sizes of the individual polymeric chains are not critical tothe action of the poloxamer, and the poloxamer can also be injected intothe blood stream or applied topically in the same manner as PEG.(Poloxamers are also amphipathic polymers to a greater or lesser extentdepending on the relative numbers of ethylene glycol and propyleneglycol groups.)

In the development of the present invention, the distribution of abiomembrane fusion agent, more particularly, PEG, in animals with spinalcord injuries was traced and it was found that PEG specifically targetsthe hemorrhagic injury in spinal cord following any means of introducingit to the blood supply (for example, parenterally such as intravenous,subcutaneous, or intraperitoneal injection, transdermally, orally,through buccal administration or via another route of administration).Furthermore, PEG appears to more uniformly bathe the injury site whendelivered by the blood supply than when it is applied to the injurydirectly. In testing the application or administration of PEG to spinalcord injured guinea pigs, it has been observed that the recovery offunctions (both in nerve impulse conduction through the spinal cordinjury and behavioral recovery) has been identical to that previouslydetermined in response to topical (direct) application of PEG to thesite of nerve tissue injury.

This is a dramatic and unexpected finding. A single dose of abiomembrane fusion agent such as PEG in aqueous solution administeredbeneath the back skin (subcutaneous injection) will reverse manyfunctional deficits in severe or traumatic spinal cord injuries inguinea pigs when the dose is administered up to six (6) to eight (8)hours post injury. The PEG migrates to and selectively attaches to thesite of a mammalian nerve tissue injury and functions there as abiomembrane fusion agent.

Tests show that the application or administration of a biomembranefusion agent such as PEG to severe spinal cord crush/contusion injuriesin situ produces functional recovery of an identified spinal cordmediated behavior in test mammals as well as a rapid recovery ofrecorded nerve impulses ascending the spinal cord through the originallesion. These physiological and behavioral recoveries following severespinal cord injury in the test mammals are not temporary but ratherstable, even improving with the passage of time. Moreover, theapplication of a biomembrane fusion agent such as PEG can be delayed forat least 8 hours after spinal cord injury without a loss in itseffectiveness.

Accordingly, the present invention contemplates a method of treatinginjured mammalian, preferably human, nerve tissue that includesadministering an effective amount of a biomembrane fusion agentexemplarily including a hydrophilic polymer such as a polyalkyleneglycol (or oxide), or block copolymers and mixtures thereof, or abiocompatible surfactant such as a nonionic amphipathic polymer (e.g., apoloxamer or a poloxamine), or mixtures thereof. Preferably, thetreatment includes an injection of the biomembrane fusion agent into apatient parenterally, including intravascularly, intramuscularly,subcutaneously, intraperitoneally, or through any other path whichresults in a delivery of the biomembrane fusion agent to the site of theinjury via the vascular system.

The present invention also contemplates the administration of aneffective amount of a biomembrane fusion agent in the form of ahydrophilic polymer such as a polyalkylene glycol or in the form of abiocompatible surfactant such as a nonionic amphipathic polymer to thesite of a nerve tissue injury by contacting the nerve tissue with aneffective amount of the biomembrane fusion agent directly applied in abath to the nerve tissue. Where the biomembrane fusion agent is apolyalkylene glycol, it can preferably and particularly take the form ofC₁ to C₁₀ polyalkylene glycol such as polymethylene glycol, polyethyleneglycol, polypropylene glycol, polybutylene glycol, polypentylene glycol,polyhexylene glycol, polyheptylene glycol, polyoctylene glycol,polynonylene glycol, and polydecylene glycol, including branched andstructural isomers thereof. The biomembrane fusion agent may moregenerally take the form of any mixture of acceptable individual agents,such as mixtures of two or more polyalkylene glycols, including branchedand structural isomers thereof, mixtures of polyalkylene glycols withblock copolymers of polyalkylene glycols, and mixtures of blockcopolymers of polyalkylene glycols. The use of polyethylene glycol,polypropylene glycol and polyethylene glycol polypropylene glycol blockcopolymers (e.g., poloxamer 188) are particularly preferred for use inthe present invention, with polyethylene glycol being most preferred. Insome applications, administration is facilitated by using a biomembranefusion agent having a reduced viscosity, e.g., reduced relative toroom-temperature viscosity by heating. Polyethylene glycol polypropyleneglycol block copolymers (e.g., poloxamer) appear to have an acceptablylow viscosity. However, it is clear that a suitably low viscosity may beattained by selecting a low-molecular-weight molecule as the biomembranefusion agent and injecting the agent after heating the agent to apermissibly elevated temperature.

In one form of the invention, a method of treating an injured mammalianspinal cord also includes directly or indirectly (by any route ofadministration including through the vascular system) administering aneffective amount of a potassium channel blocker to the site of nervetissue damage, together with an effective amount of a selectedbiomembrane fusion agent. The potassium channel blocker can be, forexample, an amino-substituted pyridine, such as 4—aminopyridine.

Yet other aspects of the invention provide compositions for treating aninjured mammalian nervous system, such as an injured mammalian spinalcord, that include effective amounts of a biomembrane fusion agent andoptionally a potassium channel blocker as described above. It has beenunexpectedly found that such compositions synergistically treat adamaged spinal cord.

Where the biomembrane fusion agent takes the form of polyethyleneglycol, it is administered in an effective amount and preferably withinthe dosage range of about 15 to 50 mg of PEG per body weight of thepatient in kilograms where the PEG has a weight of about 1500 to 4000Daltons. The fusion agent is preferably administered in combination witha pharmaceutically acceptable carrier, additive or excipient, morepreferably in a sterile injectable saline such as lactated Ringer'ssolution or any other IV “fluids” commonly administered after trauma asa treatment for shock and/or blood loss. Any polyalkylene copolymerhaving a safe clinical use as an injectable treatment in other contextsis suitable for use in a method for treating injured nerve tissue inaccordance with the present invention.

Where the fusion agent is poloxamer, a polyethylene—polypropylene—polyethylene block copolymer, or a poloxamine, it is administeredpreferably in an isotonic sterile saline such as a lactated Ringer'ssolution, USP sterile isotonic saline solution, Kreb's solutions, orother IV “fluids” solution at fusion agent dosages of 50-150 mg/kg ofthe patient's body weight, for instance, about 100 mg/kg of body weight.The aqueous solution is prepared in such a way tas the injection isapproximately 1 cc. Poloxamers are preferably accompanied by a potentantioxidant. For instance, 0.4 g of a natural antioxidant, Vitamin C,may be added to the stock solution of 350 mg/Kg P188. Any nonionicsurfactant or amphipathic polymer having a safe clinical use as aninjectable treatment in other contexts is suitable for use in a methodfor treating injured nerve tissue in accordance with the presentinvention.

The methodology of the present invention will permit a physician ormedical practitioner (e.g., neurosurgeon) to physically and functionallyreconnect transected nerve cell processes (axons), as well asimmediately rescue crushed nerve processes that would otherwise progresson to axotomy and the irreversible loss of the distal axonal segment.This result is surprising. The methodology of the present invention isunexpected and dramatic for at least four more significant reasons:

1) A biomembrane fusion agent as disclosed herein can be delivered bytuberculin syringe and a fine (26 gauge) needle inserted just under thesheath of peripheral nerves near the site of crush or stretch and/or byIV injection. This operation has been performed with PEG and poloxamerin adult guinea pigs with focal crush injuries to the sciatic nerve ofthe leg. Observations revealed very rapid recoveries (minutes to 1 hour)of nerve impulse conduction through the injury and recoveries of musclefunction in the lower leg (originally extinguished by the crush of therelevant nerve).

2) Administration of a biomembrane fusion agent through the blood supplyof a patient with injured nerve tissue relieves the attendingneurosurgeon of the absolute requirement to surgically expose the siteof the nerve tissue injury, for instance, to remove the tough coveringof the spinal cord (the dura), before a topical application of thefusion agent is made.

3) Introduction of biomembrane fusion agents through the blood supplyenormously facilitates the time in which these agents could be deliveredclinically. The fusion agents can be delivered as a component of IVfluids that are standardly begun even at the accident site minutes tohours after injury.

4) Introduction of a biomembrane fusion agent such as PEG and/orpoloxamer through the vasculature (blood supply) also enables the use ofthis therapy in cases of severe head injury, as well as cerebralhemorrhage (stroke). These traumas would not have been accessible to thetopical application and removal of fusion agent solutions, but areperfectly accessible to the treatment by IV injection through the normalIV fluids continuously delivered to trauma patients. Head injury andstroke are hemorrhagic events identical to spinal cord injury in thatcells in these regions of the brain begin to undergo dissolution anddeath after they become permeabalized by even a temporary restriction ofblood supply. The breaches in the membranes of the nerve cells can bemolecularly sealed and the cells rescued by fusion agent applicationjust as in spinal cord trauma.

An injection of a biomembrane fusion agent pursuant to the presentinvention should be made as soon as possible after a severe injury tothe central nervous system. Since the biomembrane fusion agent isdelivered via the blood stream, this methodology can be used to treatany form of traumatic damage to the peripheral nervous system (crush orinjury where nerve fibers are not completely severed), any form ofdamage to the spinal cord where the cord itself is not severed into twopieces, any type of traumatic damage to the brain such as blunt forcetrauma or concussion, and stroke or cerebral aneurysms.

It is therefore an object of the invention to provide methods andcompositions for treating a mammalian nerve tissue damage to at leastpartially restore nerve function.

These and other objects and advantages of the present invention will beapparent from the descriptions herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 14C are directed to experimental proceduresdemonstrating the effect of a direct application of a biomembrane fusionagent such as polyethylene glycol to exposed damaged nerve tissue. FIGS.15 through 23C are directed to experimental procedures demonstrating theeffect of an intravascularly delivered biomembrane fusion agent, such aspolyethylene glycol, on damaged nerve tissue.

FIGS. 1A through 14C are more specifically directed to experimentalprocedures performed to study and determine the effects of topicallyapplied PEG to in vitro and in vivo spinal cord injury in guinea pigs.

FIGS. 1A-1B depict experimental apparatuses used in the study. FIG. 1Adepicts a top view of the double sucrose recording chamber. In FIG. 1A,from left to right, the first large compartment contains 120 mM KCl, thecentral large compartment contains the physiological test solutions,such as oxygenated Krebs' solution, and the third compartment alsocontains 120 mM KCl. The small chambers on either side of the centralcompartment contain 230, mM sucrose. Seals fashioned from coverslips aresecured in place with high vacuum silicone grease at the locations shownto inhibit the exchange of the various media from one compartment to thenext. AgAgCI electrodes for recording and stimulation are in series withsocket connectors at the locations shown. In the top portion of FIG. 1B,a side view of the apparatus used to produce a standardized crush to theisolated spinal cord at its midpoint within the central compartment isshown. The position of the spinal cord injury within the central chamberis shown in the lower portion of FIG. 1B. The apparatuses are furtherdescribed below with reference to Example 1.

FIG. 2 depicts electrophysiological recordings of control andPEG-treated guinea pig spinal cords at 10 seconds and 5 minutes aftercrushing as more fully described in Example 1. Top panel:electrophysiological recordings show the compound action potentials(CAPs) before the standardized experimental crush and the immediate lossof conduction after experimental injury. Bottom panel:electrophysiological recordings show a typical response to acutestandardized injury of the isolated spinal cord strip after PEGtreatment. SA, stimulus artifact.

FIG. 3 depicts a graph showing the recovery of the CAP as a percentageof the precut amplitude as a function of time post-crush. Average CAPsand their standard error (SE) are displayed for 10 spinal cord strip foreach group.

FIGS. 4A-4D depict analyses of the CAP amplitude as a function ofincreased strength of stimulus in control and PEG-treated spinal cords.FIG. 4A depicts a series of 10 superimposed electrophysiologicalrecordings showing CAPs in response to 10 separate increasing stimulusintensities (0.015-2.0 mA, 100 μs duration squarewave stimuli) prior tothe experimental crush and 1 hour after the crush in a controlpreparation. FIG. 4B is graph showing the preinjury amplitude vs. postinjury amplitudes for 4 spinal cord strips in a modestly injured controlgroup. FIG. 4C shows a graph of preinjury amplitude vs. post-injuryamplitudes showing the hypothetical skewing of data where (a) more largecaliber—fibers (with a lower stimulus threshold) are responsible for theCAP or (c) more small caliber fibers are recruited to produce therecovered CAP following injury relative to unity (b). FIG. 4D shows theactual distribution of these data points in the PEG-treated group.

FIGS. 5A-5C depict graphical representations of refractory periodchanges in control and PEG-treated spinal cords after double pulsestimuli. In FIG. 5A, twenty individual records of CAP responses to twinpulse stimuli are superimposed. The first of these twenty stimuliproduced the single large CAP marked with the arrow. Since this firstCAP is always, produced by a stimulus of the same intensity, each ofthese superimposed individual electrical records was identical. Fromleft to right, the CAP produced by the second stimulus is shown. Notethe typical dampened amplitude of the second CAP when triggered duringthe relative refractory period followed by the typical plateau inamplitude produced when the second stimulus is applied subsequent to therelative refractory period. In FIGS. 5B and 5C, the response to thesecondary stimulus (as a % of the first CAP amplitude) vs. theinterstimulus interval is plotted for 4 untreated and PEG-treated spinalcord strips, respectively. Filled circles show data points prior to thestandardized crush injury while open circles show data points obtained 1hour after the injury.

FIGS. 6A-6B depict electrophysiological recordings showing CAPs ofcontrol, and PEG/4-AP treated spinal cords. In FIG. 6A, untreated spinalcord strips were treated with 100 μM 4-AP at 1 hour post-injury. In FIG.613, 100 μM 4-AP was administered 1 hour post-PEG application. FIG. 6Cis a bar graph of group data showing percent amplitude increase for 5control and 5 PEG-treated spinal cords.

FIG. 7 depicts a proposed mechanism of the synergistic effect of PEG and4-AP as more fully described in Example 2. The membrane lesion obtainedby mechanical compression is depicted by holes. Small arrowheadsrepresent potassium channels.

FIG. 8 depicts the laboratory device used to stabilize and hold the twosegments of the spinal cord together during fusion.

FIGS. 9A-9I depict electrophysiological recordings in adult guinea pigspinal cords at 37° C. FIG. 9A shows a normal compound action potential(CAP) recorded from a strip of ventral white matter prior to cutting thespinal cord. Two minutes after this record was taken, the strip wascompletely severed transversely, eliminating CAP conduction to therecording site as seen in FIG. 9B. Although the CAP began to recoverwithin 15 minutes of PEG application, FIG. 9C shows the weak recoveringcompound action potential 60 minutes post transection. FIG. 9D istypical of all fused cords tested, where a second transection throughthe fusion plane eliminated the recovered CAP. FIGS. 9E-H show theresults of control experiments. FIG. 9E shows a typical CAP. In FIG. 9F,this CAP was subsequently eliminated following transection when the twosegments of white matter were tightly abutted and treated identically tothe fusion procedures except that PEG was not applied. In FIG. 9G,another typical CAP is shown. After transection, the spinal cord stripsused to obtain FIG. 9G data were loosely abutted following completetransection and PEG was applied. Note the lack of any recovered CAP inFIG. 9H. FIG. 1 shows another recovered CAP produced by PEG fusion atthe same level of amplification as shown in FIG. 9C. All tracesrepresent a computer average of 20 individual records. The scale bar inFIG. 9A is for FIGS. 9A, 913, and 9D. The scale bar in FIG. 9E is forFIGS. 9E-H.

FIGS. 10A-10D show fluorographs of adjacent transected surfaces of atransected strip of guinea pig spinal cord white matter facing eachother across a gap. About 1.5 μl of FIR was injected into segment A, andrevealed using excitation/barrier wavelengths of 545/590 nm,respectively, in darkfield. The adjacent segment in B, illuminated withthe same excitation and barrier filter combination, was injected withFE. Note the absence of FIR filled axons in B. Images C and D areidentical views to A and B, only illuminated with excitation/barrierwavelengths of 495/515 nm sufficient to reveal only the FE labeled axonsseen in D. Note that in these control preparations, as in all unfusedregions of spinal cords, dye labeled axons are never visualized in thesegment of spinal cord not originally injected. The dashed lines showthe approximate boundaries of a projection of dye labeled axons fromtheir site of injection (out of the photographic field) to the plane oftransection. Scale bar for A, B, C, and D=500 μm.

FIGS. 11A-11F depict fluorographs of PEG-fused regions of four separatespinal cords strips that were initially transected, treated with PEG andinjected with flourescent dyes as more fully described in Example 4. Theapproximate plane of fusion is marked with a dashed line. An FR-labeledprojection is shown in FIG. 11A at low magnification. The rectangle inFIG. 11A circumscribes the region shown in FIG. 11B at highermagnification. The arrows in FIG. 11B depict sites where one axonsegment appeared to be fused to two others. In FIG. 11C, the arrowspoint to three of many terminal clubs of unfused fibers within the FEinjected segment of the cord mingling with fused fibers traced acrossthe original plane of transection. The arrows in FIG. 11D point to twoFIR-labeled fused axons that could be traced across to the opposite cordsegment. The asterisk (*) marks one nearby unfused axon ending in aterminal club near the transection plane. In FIG. 11E, a 1 micronplastic embedded section is shown, displaying a region of axonreattachment. FIG. 11F is a higher magnification view of a plasticsection adjacent to one shown in FIG. 11E, and shows that the site ofcontinuity is produced by a collection of abnormal, unmyelinated axonsegments which are in continuity with myelinated axons in both halves ofthe white matter strip. The scale bars are: FIG. 11A=50 μm, FIG. 11C=25μm, FIG. 11D=20 μm, FIG. 11E=10 μm, and FIG. 11F=5 μm.

FIG. 12A is a diagram of the sensory and motor components of thecutaneous trunci muscle (CTM) reflex of the guinea pig as more fullydiscussed in Example 5. FIGS. 12B-12D depict drawings of captured andsuperimposed video images of a guinea pig during a period of CTMstimulation with a monofilament probe. Two video frames weresuperimposed to show the position of the dots prior to stimulation and{fraction (1/25)}th second after stimulation [see also Blight et al.,(1990) J. Comp. Neurology 296: 614-633; and Borgens, R. B. and Shi, R.(1999) J. Faseb (in press)]. FIG. 12C shows the receptive field prior tospinal cord injury. One circumscribed region is a superimposed image 4days post injury which shows the region of CTM loss. Within this region,tactile stimulation no longer produced contraction of the skin. In thissham-treated animal, CTM functioning remained unchanged until sacrifice1 month post—injury. FIG. 12D shows behavioral recovery following PEGapplication: From left to right, the first drawing shows the normal CTMreceptive field prior to spinal cord injury. The second drawing showsthe undamaged receptive field and the region of CTM loss is shown priorto the application of PEG. The third drawing shows the same guinea pig 4days following the application of PEG. The region of CTM behavioralrecovery, which was observed within the first 6 hours post PEGapplication and which increased in size with time to restore about 29%of the area of CTM behavioral loss by 4 days post injury, is outlined.

FIG. 13A depicts an experimental setup used in the examples. Nerveimpulse pathways were interrupted by crushing the spinal cord in themidthoracic region. A control procedure demonstrated that a failure todetect SSEPs was due to a failure of ascending nerve impulse conductionthrough the lesion by stimulation of a neural circuit unaffected by theinjury.

FIGS. 13B-13D depict SSEP electrical recordings. The top panel of FIG.13B shows a complete somatosensory evoked potential (SSEP) electricalrecording in an uninjured guinea pig. The lower panel of FIG. 13B showsthe three individual traces used to produce the averaged signal seen inthe top panel. SA=stimulus artifact; P1=first arriving SSEP(latency=about 18 ms); P 2=late arriving potentials (latency=about 34ms). The arrow in the lower panel of FIG. 13B points to a typical SSEPin response to median nerve stimulation, showing interruption inconduction was due to the lesion. Below the median nerve controlresponse, an SSEP in response to tibial nerve stimulation 4 dayspost-injury is depicted. In FIG. 13C, SSEPs are shown before and afterPEG application. From top to bottom: a typical SSEP prior to spinal cordinjury; an SSEP showing immediate loss of the SSEP following injury;SSEP of a median nerve control; SSEP 1 hour post PEG; SSEP 1 day postPEG; and SSEP 4 days post-PEG. FIG. 13D depicts a graph of the mean andstandard error of both amplitude and latency of the early arriving (PI)SSEPs in 10 PEG-treated animals as a function of time after crush.

FIGS. 14A-14C depict SSEP electrical recordings in control guinea pigsand guinea pigs treated with PEG at various times postcrush. FIG. 14Ashows a typical SSEP prior to compression of the spinal cord and itselimination following injury as in FIGS. 1313-13D. FIG. 14B depicts SSEPelectrical recordings of control, sham-treated animals, after variousindicated time periods. SA=stimulus artifact; P1=first arriving SSEP(latency=about 18 ms); P 2=late arriving potentials (latency=about 34ms). FIG. 14C depicts an electrical recording showing SSEPs afterdelayed treatment with PEG.

FIG. 15 depicts a surgical exposure performed on the sciatic nerve of atest mammal and shows the branches (which are cut—see methods) of thesciatic nerve and the gastrocnemius muscle. Note the position of the twotransducers, one measuring the force of muscle contraction, the otherthe displacement of the hind paw. The relative position of the hookelectrodes stimulating the sciatic nerve proximal to its insertion onthe gastrocnemius is shown as is the placement of bipolar discelectrodes on the muscle to record the spread of APs in response tostimulation. All records are acquired simultaneously on three channelsof recording equipment, a fourth channel being used to display an eventmarker triggered by the stimulation pulse. For illustration purposesonly, the drawing is not made to scale.

FIG. 16 is a graph depicting PEG-mediated recovery of CAPs in theisolated sciatic nerve of FIG. 15. The top electrical record shows a CAPstimulated and recorded within the double sucrose gap chamber. Note itscomplete elimination after transection (second trace) and its partialrecovery following abutment of the proximal and distal segments and PEGapplication (third trace).

FIGS. 17A-17C are series of graphs showing physiological measurements ofgastrocnemius activity in response to stimulation of the sciatic nervein a sham-treated animal pursuant to the experimental setup of FIG. 15.Electrical recordings of three measures of response to stimulation areshown in each of the three blocks: the event marker is shown as a dashedline. The time base shown in the top block of recordings is the same forall three sets of graphs. The scale units on the x axis are the same forall recordings unless noted. For example, the sensitivity of APrecording is increased by a factor of 2 and 10 in the lower recordingsof AP and force, respectively. It is to be noted that the threemeasurable responses to sciatic stimulation are completely eliminatedimmediately subsequent to a crush injury of the nerve proximal itsinsertion on the muscle. It is to be noted as well that this lack ofresponse is stable for the next hour—even at an increased sensitivity ofrecording.

FIGS. 18A and 18B are graphs showing physiological responses of thegastrocnemius muscle to stimulation prior to PEG treatment of a sciaticnerve injury made in the experimental procedure shown in FIG. 15. Theconventions are the same as detailed in FIG. 17A-17C. Note the 10 and100 fold increase in sensitivity of recording in AP and Forceimmediately following crush injury to the sciatic nerve and theinability to measure any response at this time or sensitivity.

FIG. 19 is a series of graphs showing recovery of functions in thegastrocnemius muscle in response to PEG treatment. This set of recordswas obtained 5 minutes after PEG treatment to the nerve injury in thesame animal whose records are shown in FIGS. 18A and 18B. Note the rapidand robust recovery of AP and muscle contractile force as emphasized bythe substantial reduction in amplifier gain required to record them.Compare recording scales to pre and post injury records shown in FIGS.18A and 18B. This recovery was stable for the next hour of monitoring.

FIGS. 20A-20C are graphs showing long term recovery of functions in aPEG-treated animal. The conventions are the same as in previous figures.These records show the loss and recovery of all three functionalmeasures subsequent to PEG treatment by one hour post injury. Note thata miniscule AP at the limit of detection may have been recorded by anincrease in amplification of 10 fold in the post-crush record. This mayhave signaled a slightly less severe injury than produced in other casesallowing a more complete response to PEG. This was the only case whereall three functions had measurably recovered subsequent to nerve injuryin any animal.

FIGS. 21A-21D are four photographic representations showing polyethyleneglycol labeling in crushed guinea pig spinal cord. In FIGS. 21A-21D, thedistribution of FI-PEG in crushed spinal cord is shown using three typesof application. The application of PEG was made within {fraction (1/2)}hour of the constant displacement crush injury, and evaluated byfluorescent microscopy of 50 μm thick frozen cross sections about 24hours later. In FIG. 21A, a typical control section is shown indarkfield—the image digitally enhanced to reveal the very faintlylabeled spinal cord. Such uninjured control sections were obtained byharvesting a segment of the spinal cord at least 3-4 vertebral segmentsfrom the injury site. Note the characteristic labeling of PEG inuninjured spinal cord at the level of detection. The arrows point toweakly labeled regions of vasculature in the gray matter and at the pialsurface. FIG. 21B shows strong labeling of PEG at the epicenter of thecrush produced by a 2-minute topical application of PEG to the lesion asin previous reports. Arrows point to relatively unlabeled centralregions of this injury. In FIGS. 21C and 21D, heavy FI-PEG labeling isshown associated with subcutaneous and intravenous injectionrespectively. In FIG. 21C, the arrow points to a cyst forming around theswollen central canal. Note the extensive labeling of only the injurysite by all methods. The scale bar=500 μm

FIGS. 22A and 22B are graphs of electrical records showing loss andrecovery of conduction in crushed guinea-pig sciatic nerves afteradministration of PEG. The first electrical record at the top of bothFIGS. 22A and 22B shows a typical SSEP recording in response to tibialnerve stimulation. Note the early and late arriving evoked potentials(P1 and P2) in the intact spinal cord, and their immediate eliminationby the spinal cord injury. Though not shown for every record, the mediannerve control procedure was performed any time an SSEP was not recorded,demonstrating the failure to record CAPs was due to the injury. In FIG.22A, a typical set of records is shown for one control animal to the 1month time point when the study was concluded. Note the complete lack ofSSEP conduction and the robust Median nerve induced SSEP. In FIG. 22B, atypical set of electrical records for a PEG-treated animal is shown.Note the elimination of the tibial nerve derived SSEP by the spinal cordinjury, and the positive median nerve control procedure performed at thesame recording time. Before the end of the first day post-injury, SSEPconduction was restored by this subcutaneous PEG injection made 6 hoursafter the injury. Recovered evoked potentials continued to improve inamplitude and latency during the next month of observation, and in nocase were recovered SSEPs lost after their recovery. The insert displaysthe amplitude and time base for all records except median nervestimulations, which were recorded at ½ this sensitivity, but using thesame time base.

FIGS. 23A-23C are tracings of captured and superimposed video images ofa guinea pig during a period of CTM stimulation with a monofilamentprobe, showing behavioral recovery following subcutaneous PEGadministration. These tracings are derived from stop motion videotapeanalysis of cutaneous trunchi muscle (CTM) stimulation regimens in whichthe entire CTM receptive field is first determined in the uninjuredguinea pig (circumscribed). Probing inside this region of back skin witha monofilament probe produces back skin contractions, while probingoutside the region does not. This line is drawn on the shaved back ofthe sedate animal with a marker while the investigator probes theregion. The entire procedure is videotaped from above, and the variousregions of both intact receptive fields and areflexia are reconstructedfrom these video images. Note that in all animals, the midthoracicspinal cord injury eliminates CTM responsiveness below the level of theinjury on both sides (circumscribed). In control animals (FIG. 23A),this region of areflexia remained unchanged for the duration of theexperiment. In PEG-treated animals (FIG. 23B), a variable region of thelost receptive CTM fields recovered within a short time of treatment.That region shows a region of CTM recovery for this one animalcomprising about 55% of the original area of CTM loss. The inset (FIG.23C) shows the 4-week video image which was used to reconstruct theregions of intact and nonfunctional receptive fields. The dot matrixallows precise alignment and superimposing of receptive fields, as wellas a deeper analysis of the vector of skin movement, the velocity ofskin contraction and latency when required (data not shown).

FIGS. 24A-24C are two tracings, on different scales, of captured andsuperimposed video images of a guinea pig during a period of CTMstimulation with a monofilament probe, showing a dot matrix evaluationof the CTM reflex. In FIG. 24A, an overlay drawing of superimposed videoimages of a guinea pig is shown. The upper dot of each pair ofjuxtaposed dots is a permanent marker tattooed on the shaved backskin,and the lower dot of each pair reveals the position of the markers atthe peak of skin contraction—captured by stop frame video analysis. Theexact place of tactile stimulation producing these CTM contractions isshown by the position of the monofilament probe MP used to stimulate theskin. During the period of testing, a boundary line BL was drawn ontothe back of the animal with a marker revealing the total CTM receptivefield. Stimulation within the circumscribed area produced skincontractions—outside it did not. The actual video image source of thedrawing is shown in FIG. 24C. The box in FIG. 24A is magnified in FIG.24B, showing that the direction of skin contraction is generally towardsthe probe MP (arrow), and shows the distance of that contraction(hatched lines). That distance (2 mm) divided by the duration in timerequired to produce it (0.12 seconds) equals the velocity of skincontraction (16.7 mm/sec).

FIGS. 25A and 25B are graphs of measured somatossensory potentials SSEP.In a preinjury record (FIG. 25A), the bottom three overlapping traceswere produced by three separate sets of standard stimulations (seemethods of Example 9 below). These signals were averaged to produce thesingle top trace revealing two peaks of early and late arriving evokedpotentials (P1 and P2) at the brain (see methods of Example 9). Thepeaks shown are characteristic SSEPs subsequent to tibial nervestimulation in adult guinea pigs. The double headed arrow shows thestimulus artifacts. A similar recording is shown in FIG. 25B; however,this record was taken within 30 minutes of a standardized compression tothe mid thoracic spinal cord. Note the complete loss of all ascendingSSEPs. In FIG. 25C, a single averaged trace is shown of a median nervestimulation control procedure recorded in this same animal withinminutes of the traces shown in FIG. 25B.

FIGS. 26A and 26B show loss and recovery of CTM receptive fields in thetests of Example 9. FIG. 26A is a drawing or tracing showing a normaland complete receptive field RF on a control guinea pig as shown inFIGS. 24A-24C and described in below with reference to Example 9. Theregion of areflexia RA is outlined on the next image produced in FIG.26A, 24 hours post injury to the spinal cord. Note that about ½ of thetotal CTM receptive field is lost due to severe spinal cord compression.One month later, this region remains unchanged. The video image used toproduce the last drawing is shown on the far right. FIG. 26B shows, fromleft to right a similar set of images to those of FIG. 26A. The normalreceptive field RF and the region of areflexia RF post spinal cordinjury are marked. The region of CTM recovery RCR in response to PEGapplication is shown in the last drawing—demonstrating a recoveredregion of CTM sensitivity comprising about 42% of the original region ofareflexia.

FIGS. 27A and 27B depict a dot matrix evaluation of a recovered CTMreflex in a PEG-treated animal per Example 9 discussed below. FIG. 27Awas made pursuant to the methods used in generating FIGS. 24A and 24B.Note however the modest region of CTM recovery RCR—revealed by receptivefield testing as described in the methods of Example 9 and in FIGS.24A-24C. In the magnified section shown in FIG. 24B, note that positionof the probe MP within the region of recovery, and the movement of theskin marker dots MD towards the probe MP. Probing outside the region RCRdid not produce CTM reflex movements, but did elicit contractions whenprobing in the region MG (above the level of the injury). FIG. 27C showsthe actual video images collapsed in layers to produce FIGS. 27A and27B.

FIGS. 28A and 28B are graphs of evoked potentials measured in the testof Example 9. FIG. 28A shows, from top to bottom, averaged traces ofevoked potentials obtained from the same animal—from the preinjuryelectrical record to records obtained 1 month post injury. Note thecomplete lack of SSEPs in response to spinal cord injury. This wascharacteristic in 100% of the control population at all time pointstested. A median nerve control stimulation was also carried out at thesetimes, however only the 1 month record is shown in the last trace, thearrow pointing to a strong early arriving evoked potential (refer toFIGS. 25A-25C. In FIG. 28B, a recovery of SSEP conduction in a PEGtreated animal is shown. Note the characteristic double SSEP peaks inthe uninjured animal. Note the complete loss of these peaks followinginjury and the positive median nerve control procedure carried out atthis same time point. One day post injury to 1 month post injury recordsshow the recovery of SSEP conduction. The dotted line marked theapproximate peak magnitude of the early arriving SSEP, note the latencyto peak contraction is reduced with time. Such recovering SSEPs werecharacteristic of 100% of the PEG treated animals contrasted to thecomplete lack of such conduction in all control animals.

FIG. 29 is a bar graph showing amplitude and latency of recovered SSEPSin PEG treated animals per Example 9. The peak normalized amplitude ofthe early arriving SSEP is plotted for all time points as is the averagelatency (each represented as 100% for the preinjury histogram). Notethat the average magnitude of the SSEP vacillates around about 50% ofits preinjury level, while the latency incrementally declines. Thelatency at 1 month was statistically significantly reduced compared today 1 measurements. The error bars are standard errors of the mean.Measurements from 13 animals total are shown for all points except at 2weeks, where 9 animals were used for recordings.

FIGS. 30A-30D depict a portion of a neurological examination for outcomemeasures and recovery from paraplegia. A dog is placed on its side whilea neurologist tests for the presence of superficial pain (A), deep pain(B), and conscious proprioception (C and D). Skin of the flank and limbswas pinched sharply with hemostats probing for a reaction from thesubject during tests of superficial pain response. Deep pain responsewas similarly determined, but by a sustained and sharp squeeze of thejoints of the digits. Positive responses were provided for comparison bytesting the fore limbs. The responses were quantified by a 1-5 score:1=no detectable response; 2=a response at the limits of detection,indicated by an increased state of arousal, increased respiration orpulse; 3=consistent attention to the painful stimulus but without anyovert defensive behavior; 4=mildly defensive behavior such as abruptturning of the head towards the stimulus, and whining; 5=completelynormal response to painful stimuli including yelping, biting, andaggressive behavior. These scores were obtained for both sides of thebody and averaged. Conscious proprioceptive placing (CP) and weightsupport was tested in dogs by providing lateral support of the hindlimbs, and turning one hind paw “under” so that the dorsal surface ofthe paw (and the animal's weight) rested on the table (inset C). Anormal animal briskly replaces the paw to a normal stance instantlyafter the examiner releases the paw. Paraplegic animals rest in this“knuckled under” stance for extended periods of time. Testing the foreleg provided a positive control. The test was performed on each side ofthe body, and scored on each side: 1 point=complete absence of CP, and2.5 points for a positive CP response. These scores were then summed foreach animal. Voluntary locomotion (not shown) was evaluated with asimilar 1-5 point score: 1=complete inability to step or voluntaryambulate; 2=stepping and load bearing at the limit of detection, at besta few steps before falling (paresis); 3=longer sequences of stepping,poorly coordinated before falling (paresis), and unable to climb stairs;4=more robust and effective walking but with clear deficits incoordination, effective weight support, but able to climb stairs;5=completely normal voluntary walking, indistinguishable from a normalanimal. All neurological exams were videotaped for reference and halfpoints were permitted at the examiner's discretion. A total neurologicalscore (TNS) was determined for each animal at each testing period bysumming the scores of these 4 independent tests. Thus the range of apossible score for any one animal was 4 (a totally paraplegic animal) to20 (a totally normal animal, indistinguishable from an uninjured one).

FIG. 30E shows a comparison of control and PEG-treated animals (FIG.30A-30D) for each of the four outcome measures at approximately 3 dayspost injury (about 48 hours after the last PEG injection), 1 week, and6-8 weeks post injury. The y-axis for each bar graph is the percentageof the population (i.e., 25, 50, 75%). DP=deep pain, SP=superficialpain, P=proprioceptive placing, and L=voluntary locomotion. Asterisksnote when a test for proportions (Fisher's exact test, two tailed) or acomparison of the means (Students T, or the Welch variation) revealedstatistical significance. Note the clear recovery of outcome measureswithin 48 hours of the last PEG injection in that group, and thestriking improvement in TNSs in PEG-treated dogs at every period ofevaluation.

FIG. 31A shows a sedated dog and electrode placement inelectrophysiological tests for conduction through a spinal cord injuryto determine a Somatosensory Evoked Potential (SSEP). At eachevaluation, four to seven sets of evoked potentials (SSEPs) werestimulated, recorded, averaged, and stored using a Nihon KohdenME#B-5304K 4 Neuropak recorder. More particularly, FIG. 31A shows thesedated dog and the placement of bipolar stimulating pin electrodes,inserted subcutaneously, in the hind limb at the distal popliteal areaapproximately 0.5-1 cm apart. These electrodes stimulated the tibialnerve of the hind limb. A similar procedure was used to stimulate themedian nerve of the forelimb. Trains of square wave stimulations(0.5-3.0 mA amplitude, 200/min) were applied to evoke compound nerveimpulses from these nerves. To record evoked potentials, scalp needleelectrodes were inserted subcutaneously over the somatosensory cortexcontralateral to the side stimulated, while reference electrodes wereinserted on the opposite side between the mastoid and the pinna of theear. The placement of recording electrodes was facilitated bystimulation of the median nerve at the outset, a neural circuit above,and unaffected by, the spinal cord injury (inset, circuit 2). Thisprocedure also provided a positive control recording to validate thefrequent inability to record evoked potentials stimulated at the hindlimb—but whose ascending potentials are blocked by the spinal cordlesion (inset, circuit 1).

FIG. 31B is a graph of a complete set of SSEP recordings from theprocedure of FIG. 31A. A lower group of waveforms in this pair are thethree individual trains of 200 stimulations as discussed, and an upperwaveform is the averaged evoked SSEP (only such averaged SSEPs areprovided in subsequent records, FIGS. 32A and 32B). This record is of acontrol procedure. Note the clear evoked potential, recordedapproximately 10 ms after stimulation of the median nerve.

FIG. 31C is a graph showing a portion of an electrical recording,displaying three trains of stimulation, as well as the averaged SSEP asin FIG. 31B. This record was in response to stimulation of the tibialnerve in the same paraplegic dog providing the record in FIG. 31B,approximately 4 days post-injury. The complete elimination of SSEPconduction through the lesion is characteristic of all neurologicallycomplete paraplegic animals meeting the criteria described in the text,both in this and all previous reports using identical procedures (R. B.Borgens et al., J. Restorative Neurology and Neurosci. 5, 305 (1993); R.B. Borgens et al., J. Neurotrauma 16, 639 (1999)). SA=stimulus artifact;time base=50 msec full screen, 5 msec/div; sensitivity=1.25 μV/div.

FIGS. 32A and 32B relate to PEG induced recovery of nerve impulseconduction through the site of spinal injury. In FIG. 32A, a 6-weekprogression of recovery of conduction through the lesion is shown for aPEG-treated dog. Each trace is the averaged waveforms of 3-4 trains of200 stimulations as described in FIGS. 30A-30E. There is completeabsence of an SSEP in this paraplegic animal prior to surgery, andapproximately 4 days later. The third trace is a median nerve controlprocedure. There is no evidence of recovered conduction at 1 week postinjury. By 6 weeks post-surgery, two distinct evoked cortical potentialshad returned, a typical early arriving peak of approximately 26 mseclatency (P 1), and a later arriving peak (P 2), of approximately 45 mseclatency.

In FIG. 32B, a low amplitude, long duration, but reproducible evokedpotential recovered within 15 min of a slow injection of PEG is shown.This atypical SSEP appeared to segregate into an early arriving peak ofabout 15-20 msec latency, and a more condensed and later arriving peak(P 2) of about 32-35 msec latency. SA=stimulus artifact. The time baseand sensitivity scale is for both FIG. 32A and FIG. 32B.

Definitions

The term “nerve tissue” as used herein refers to any vertebrate nervetissue, particularly including cells of the central nervous system (CNS)and peripheral nervous system. More particularly, nerve tissue includesspinal cord neuronal structures, peripheral nervous system nerves, andnerve cells of the brain.

The word “injury” is used herein to generally denote a breakdown of themembrane of a nerve cell, such that there is a collapse in the abilityof the nerve membrane to separate the salty gel on their insides(cytoplasm) from the salty fluid bathing them (extracellular fluid). Thetypes of salts in these two fluid compartments is very different and theexchange of ions and water caused by injury leads to the inability ofthe nerve to produce and propagate nerve impulses—and further to thedeath of the cell. The injury is generally a structural, physical ormechanical impairment and may be caused by physical impact, as in thecase of a crushing, compression, or stretching of nerve fibers.Alternatively, the cell membrane may be destroyed by or degraded by achemical imbalance or physiological malfunction such as anoxia (e.g.,stroke), aneurysm or reperfusion. In any event, an “injury” as that termis used herein more specifically contemplates a nerve membrane defect,interruption, breach, or rupture (in the phospholipid bilayer) which canbe treated and sealed by the administration of a biomembrane fusionagent as described herein.

The term “biomembrane fusion agent” is used herein to designate any andall molecules which are not only compatible with vertebrate, and morespecifically mammalian, nerve cells but also have an affinity for nervecell membranes so as to attach to injured nerve cells at the site of aninjury. A biomembrane fusion agent thus serves in part as a kind ofbiological cement or filling material which bridges over ruptures inneuronal structures. This sealing is extremely rapid (minutes) andfacilitates the repair of the damaged neuronal structures by naturalphysiological processes which are complete at much later times (1-7hours). The sealing of neuronal membranes as described herein naturallyarrests or inhibits the progressive destruction of nervous tissue afteran injury to the nerve cell. Exemplary biomembrane fusion agents includehydrophilic polymers such as polyalkylene glycols (polyalkylene oxides)and polyalkylene glycol block copolymers such as polyethyleneglycol/polypropylene glycol block copolymers (e.g., poloxamer 188) andethylene oxide-propylene oxide-ethylene oxide (EPAN), and furtherinclude biocompatible surfactants, particularly nonionic surfactants andmore particularly amphipathic polymers such as poloxamines. Poloxamersmay also be considered to be amphipathic polymers. Poloxamers arehydrophilic to the extent that there is a greater number or greaterweight percentage of ethylene glycol groups as opposed to propyleneglycol groups. A biomembrane fusion agent at that term is used hereinmay comprise a collection, mixture, or combination of individualbiomembrane fusion agents each of which is effective in its own right toseal ruptures in nerve membranes.

The term “effective amount” when used herein with reference to abiomembrane fusion agent denotes a quantity of the agent which, whenadministered to a patient or subject, is sufficient to result in ameasurable improvement in electrical and/or behavioral function of anerve which has been so damaged or injured that normal functioning isnot possible. As discussed below, the efficacy of the treatment may bedetermined in a variety of ways, including methods which detectrestoration of nerve function. With respect to the use of the term“effective amount” with other agents, for example, potassium channelblockers, that term is used to describe an amount of an agent effectivewithin the context of that agent's use in the present invention.

The term “hydrophilic polymer” means any macromolecule (molecularweights of 200 daltons and greater) which exhibits an affinity for orattraction to water molecules and which comprises multiple instances ofan identical subunit (“monomer”) connected to each other in chainedand/or branched structures.

A “surfactant” is a molecule exhibiting both an affinity for orattraction to polar molecules such as water and an affinity for orattraction to non-polar molecules such as lipids, fats, oils, andgreases. A “nonionic surfactant” is electrically neutral, i.e., carriesno positive or negative charge. However, a nonionic surfactant may havelocalized quantum variations in charge leading, for example, to a polarsubstructure evidencing an affinity for other polar molecular structuressuch as water molecules. In the context of the present disclosure,surfactants include amphipathic polymers.

An “amphipathic polymer” as that term is used herein relates to polymerswhich have localized quantum variations in charge giving rise to polarsubstructures and non-polar substructures. The polar substructuresevidence an affinity for or attraction to other polar molecularstructures such as water molecules (hydrophilic), while the nonpolarsubstructures exhibit an affinity or attraction for nonpolar moleculessuch as lipids, oils, greases, fats, etc. (lipophilic).

Poloxamers, also called non-ionic detergents, and/or triblock polymers,comprise a polyethylene glycol chain(s) (block 1), then a polypropyleneglycol chain (block 2), followed by a polyethylene glycol chain(s)(block 3). These compounds can be synthesized in numerous conformationsand molecular weights. The weights of the various “blocks” can even varybetween themselves—leading to a complicated nomenclature. What all ofthe poloxamers have in common is a hydrophobic head group (block 2),surrounded by hydrophilic (PEG) chains. The hydrophobic “head” isbelieved to insert itself into the “hole” in a membrane (where thehydrophobic interior of the bilamminer membrane is exposed) while thehydrophilic PEG arms interdigitate and link with or attach to thenearby, more normal, membrane.

The term “poloxamine” denotes polyalkoxylated symmetrical block polymersof ethylene diamine conforming to the general type[(PEG)_(x)-(PPG)_(y)]₂-NCH₂CH₂N-[(PPG)_(y)—(PEG)_(x)]₂.

The word “biocompatible” means that a substance can be placed intointimate contact with biological structures, including cells andcellular membranes, without detriment to the continued physiologicalfunctioning of the contacted cells and membranes.

The term “polyalkylene glycol” refers to a molecule having the chemicalformula H(O[CH₂]_(m))_(n)OH where m and n are nonzero integers. Theinteger m has the following values for exemplary polyalkylene glycols:polymethylene glycol (m=1), polyethylene glycol (m=2), polypropyleneglycol (m=3), polybutylene glycol (m=4), polypentylene glycol (m=5),polyhexylene glycol (m=6), polyheptylene glycol (M=7), polyoctyleneglycol (m=8), polynonylene glycol (m=9), and polydecylene glycol (m=10),including branched and structural isomers thereof. Pursuant to thepresent disclosure, polyalkylene glycols have a molecular weight betweenabout 200 and about 25,000 daltons, and preferably between about 400daltons and about 3500 daltons.

The word “carrier” is used herein to denote a liquid matrix, medium orsolvent in which molecules of a biomembrane fusion agent are dispersedor distributed. A pharmaceutically acceptable carrier is one which isbiocompatible to vertebrate and more particularly mammalian tissues.Generally acceptable carriers include water, saline solutions, amongnumerous others.

By definition a “potassium channel blocker” or “K⁺ channel blocker” isany agent that specifically and sterically inserts itself into (orotherwise deactivates) any of the several and growing classes of K⁺channels. This includes both fast and slowly activating channels andboth “voltage gated or non-gated” channels. Almost all channels for K⁺are “gated” by the voltage across the cell membrane. When these channelsare open, K⁺ tends to move from the cytoplasm into the extracellularfluid because it is about 100 times more concentrated inside thanoutside the cell. This K⁺ exodus (which among other things helpsextinguish the nerve impulse, bringing the membrane potential back to aresting state) can thus be “blocked”. In regions of demyelination ormembrane potential polarization, K⁺ channel blockade can both increaseexcitability, as well a extend the distance along a nerve fiber in whicha nerve impulse can travel before it is extinguished. In spinal cordinjury, this may only be a few millimeters of nerve fiber damage, withabsolutely normal membrane on either side. There are many known K⁺channel blockers including reversible blockers (TEA) and some proteins(synthesized from snake venoms) that irreversibly block these channels.Potassium channel blockers include substituted pyridines and, moreparticularly, amino-substituted pyridines. The application of K⁺ channelblockers to spinal cord repair as described herein involves the fastpotassium channel, type 1, blocker 4-AP (4-aminopyridine) and its analog3, 4 di-aminopyridine. Too high a dosage, or the use of the otherblockers (more non specific and poorly reversible) may lead toconvulsions and even death.

The delivery of a biomembrane fusion agent via a vascular system of apatient entails the administration of a biomembrane fusion agent via apathway including one or more veins and/or arteries of the patient.Instead of direct application in which the agent is injected into thepatient at the site of exposed nerve tissue, thevascular-system-mediated delivery of a biomembrane fusion agentcontemplates an administration and subsequent conveyance of the agent tothe site of an injured nerve via the vascular system of the patient. Theadministration of the biomembrane fusion agent is preferably byinjection, for example, via a hypodermic needle or catheterization,either directly into a vein or artery or indirectly by subcutaneousinjection into muscle tissue or intraperitoneally. Other methods mayalso be effective, for example, by ingestion, transmembrane delivery(including transdermal delivery), by suppository, through inhalants,buccally, or by implantation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to preferred embodiments andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alterations and furthermodifications of the invention, and such further applications of theprinciples of the invention as illustrated herein, being contemplated aswould normally occur to one skilled in the art to which the inventionrelates.

The present invention provides methods and compositions for treatinginjured nerve tissue of a vertebrate. The methods and compositions aredesigned to at least partially restore nerve function in the vertebrate.In one aspect of the invention, methods are provided for treating aninjured or damaged vertebrate spinal cord that include contacting thespinal cord with an effective amount of a biomembrane fusion agent. Thecompositions include a biomembrane fusion agent, preferably apolyalkylene glycol such as polyethylene glycol (chemical formula:H(OCH₂CH₂)_(n)OH) and/or a nonionic surfactant such as an amphipathicpolymer (e.g., a poloxamer or a poloxamine), and/or mixtures orcopolymers thereof. In alternative embodiments, the method may includetreating the nervous system with a potassium channel blocker, preferablya substituted pyridine, such as an amino-substituted pyridine, eitherbefore, during or after contacting the spinal cord with the biomembranefusion agent. Other aspects of the invention provide compositions fortreating an injured nervous system of a vertebrate. The preferredcompositions include a biomembrane fusion agent and a potassium channelblocker.

As indicated above, in a first aspect of the invention, a method oftreating an injured spinal cord of a vertebrate is provided. The methodis preferably performed in vivo, although it may also be used in vitro,for example, in the study of spinal cord components or functionality.

The preferred biomembrane fusion agent is a polyalkylene glycol. A widevariety of polyalkylene glycols may be used, including those, forexample, where the alkylene group is methylene, ethylene, propylene,butylene, pentylene, hexylene, heptylene, octylene, nonylene, anddecylene, including branched and structural isomers thereof. Preferably,the polyalkylene glycol will be water-soluble and is selected from thegroup consisting of polyethylene glycol, polypropylene glycol and blockcopolymers of polyethylene glycol and polypropylene glycol. A morepreferred polyalkylene glycol is polyethylene glycol. Although a widerange of molecular weight polyalkylene glycols may be used (betweenabout 200 daltons and about 25,000 daltons) depending on the ability ofthe polyalkylene glycol to pass through various biological barriers suchas the digestive tract, polyalkylene glycols and polyalkylene glycolblock copolymers of molecular weight of about 400 to about 3500 daltonsare preferred. Such biomembrane fusion agents may be synthesized bymethods known to the art or may be purchased commercially.

The biomembrane fusion agent may also be a polyalkylene glycol/proteinconjugate as known in the art, wherein the protein preferably aids inscavenging free radicals. For example, the biomembrane fusion agent,such as polyethylene glycol or other alkylene oxide, may be conjugatedto catalase to form PEG-catalase, or to superoxide dismutase to formPEG-SOD. Such conjugates are available commercially from Sigma, St.Louis, Mo. The biomembrane fusion agent, may also be conjugated to abiodegradable surgical glue, such as a commercial fibrin glue, tofacilitate and stabilize reattachment and fusion of severed nervoustissue.

Alternatively, the biomembrane fusion agent may be a biocompatiblesurfactant, preferably a nonionic surfactant and more preferably anamphipathic polymer such as a poloxamer or a poloxamine.

The biomembrane fusion agent may be provided in a pharmaceuticallyacceptable carrier. Such carriers include, for example, water,preferably sterile and including distilled water, and any otherpharmaceutically acceptable carrier known to the art that will not havean adverse effect on the treatment. Sterile distilled water is apreferred carrier in work to date.

The biomembrane fusion agent is administered to the patient as soonafter injury as possible and prior to irreversible dissolution of axonalmembranes and the myelin sheath. Although this time period may varydepending on the nature and extent of the injury, the fusion agent istypically administered immediately after the injury occurs, andpreferably not later than about 24 hours post-injury, but is typicallyadministered between about 1 hour to about 8 hours post-injury. Thoughearly treatment is preferred, administration of the biomembrane fusionagent may still be beneficial for up to 2 weeks after the initial nerveinjury (called the “primary injury”). This is because nerve injury is acontinuous, slow, progressive event, especially in spinal cord where itis called “secondary injury” (Tator and Fehlings 1991, J. Neurosurgery75: 15-26).

The biomembrane fusion agent may be delivered to the site of injury byany suitable method. Preferably, the biomembrane fusion agent isadministered through the vascular system of the subject or patient. Thefusion agent may be injected directly into the vascular system orindirectly by injection intramuscularly, subcutaneously orintraperitoneally. It has been discovered that an indirectadministration of a biomembrane fusion agent such as polyethylene glycolvia the vascular system of the patient unexpectedly results in aselective adherence of the fusion agent (e.g., PEG, poloxamer or otheragent) to the injured nerve tissue. There is little or no adherence toundamaged nerve tissue. Without being limited by way of theory, it isbelieved that by adhering to damaged nerve tissue, the biomembranefusion agent promotes the natural healing processes of the damaged nervecells.

Where the biomembrane fusion agent is a polyalkylene glycol such as PEG,the fusion solution comprises fusion agent in an amount of typicallyabout 15 to about 50% by weight and preferably is administered in dosesof about 15-50 mg PEG per body weight in kilograms of the patient wherethe PEG has a weight of 1500-4000 Daltons. Where the biomembrane fusionagent is an amphipathic polymer such as a poloxamer or a poloxamine, thefusion solution typically contains fusion agent in an amount of about 15to about 50% by weight and is administered in dosages of about 15-150 mgpoloxamer or poloxamine per body weight in kilograms of the patient.

Where the agent is applied directly to damaged nerve tissue which hasbeen exposed, for example, via surgical procedures, the agent may beapplied with any suitable liquid dispensing device. Although thepercentage by weight of the fusion agent in the direct-applicationcomposition may vary, the composition typically includes fusion agent inan amount of at least about 40% by weight, more preferably about 40% toabout 50% by weight, and most preferably about 50% to about 55% byweight.

In the case of a direct-contact application, the injured site is exposedto the fusion agent for a time period effective for treating the injury.This time may vary depending on the size of the lesion, the extent andnature of the injury, the biomembrane fusion agent used, and theconcentration of the biomembrane fusion agent. The lesion is typicallyexposed to the agent for at least about one minute and more preferablyat least about 2 minutes. In preferred embodiments, the fusion agent isremoved from the injured tissue being treated prior to the occurrence ofdeleterious tissue changes. In a further preferred embodiment, theinjured tissue is exposed to the fusion agent for no more than about 5minutes. After the injured region of the nervous system is treated withthe fusion agent, it may be removed by aspiration and the treated sitewashed with a biowashing solution, such as isotonic Kreb's solution asdescribed in the examples. Excess fusion agent and/or Kreb's solutioncan then be removed by aspiration.

In another form of the invention, the method may further includeadministering to the patient or subject an effective amount of apotassium channel blocker. In the case of a direct-contact applicationof a biomembrane fusion agent, the injured site is contacted with aneffective amount of a potassium channel blocker in addition to thebiomembrane fusion agent. A variety of potassium channel blockers may beused, including substituted pyridines. Preferred potassium channelblockers include those that improve action potential conduction ininjured tissue, including 3,4-diaminopyridine, 4-methylaminopyridine andampidine. In a preferred form of the invention, the pyridine issubstituted with an amino group, more preferably at the 4-position ofthe ring. Moreover, it has unexpectedly been discovered that treatmentof an injured mammalian spinal cord with a potassium channel blocker,such as 4-aminopyridine, after treatment with a fusion agent, such aspolyethylene glycol, can result in synergistic repair of the spinalcord. For example, compound action potentials (CAPs) increase inconduction when both agents are used by a percentage greater than thesum of the percent increase in conduction of the CAPs when injuredspinal cords are treated alone with either the fusion agent or thepotassium channel blocker.

Although the injured nervous system may be contacted with the potassiumchannel blocker prior to or at the same time as treating with the fusionagent, the system is preferably contacted with the blocker after thetreatment with the fusion agent. The potassium channel blocker may beapplied in a fashion similar to the fusion agent. The amount of thepotassium channel blocker effective in treating or repairing the injurednervous system, such as injured mammalian spinal cord, will alsosimilarly depend on the factors mentioned above. When the potassiumchannel blocker is 4-aminopyridine, it is typically applied at aconcentration of about 10-100 ng/ml cerebrospinal fluid and furtherpreferably about 50-100 ng/ml cerebrospinal fluid. After treatment with4-aminopyridine, it can similarly be removed by aspiration and thelesion site washed with the biowashing agent.

In yet other forms of the invention, the method may include treating theinjury with a polyalkylene glycol, as well as with other conventionalmanagement compounds and/or compositions. For example, in addition totreatment with a polyalkylene glycol, the injury may be treated with asteroid, such as methylprednisolone.

A wide variety of injuries may be treated in the present invention. Invarious forms of the invention, the injury may arise from a compressionor other contusion of the spinal cord, crushing of the spinal cord orsevering of the spinal cord, or anoxia (e.g., stroke), aneurysm orreperfusion.

The efficacy of the treatment may be determined in a variety of ways,including methods which detect restoration of nerve function. Forexample, restoration or increase in conduction of action potentials,such as CAPs, through the injured site may be used as an indicator thatnerve function has at least partially been restored as described in theexamples. Nerve function is considered to have been at least partiallyrestored if there is an increase in the conduction of action potentialsafter treatment. Preferably, the treatment will be conductedsufficiently to achieve at least about 10% increase in conduction ofCAPs. Moreover, restoration of anatomical continuity may also beobserved by examination with high-resolution light microscopy and/or bydiffusion of intracellular fluorescent dyes through the repaired nervoustissue, such as repaired axons, or by direct observation of repairedaxonal membranes. Additionally, in human applications, the efficacy ofpreferred treatments may be observed by the restoration of more than onespinal root level as determined by the American Spinal InjuryAssociation (ASIA) motor score and/or the National Animal Spinal CordInjury Study (NASCIS) score as know in the art and as described in Wagihet al., (1996) Spine 21: 614-619. Furthermore, in veterinaryapplications, behavioral analysis of the cutaneous trunci muscle (CTM)reflex, as more fully discussed in the examples, may also be used todetermine the efficacy of the treatment, and whether nerve function hasat least partially been restored. Using this analysis, nerve function isconsidered to have been at least partially restored if there is anincreased reflex behavior after treatment, but treatments are desirablypreferred so as to achieve at least about a 10% increase in the area ofCTM behavioral recovery.

In yet other aspects of the invention, compositions for treating aninjured nervous system of a vertebrate are provided. The compositionsare designed to at least partially restore nerve function as describedbelow. In one form, a composition includes a biomembrane fusion agentand a potassium channel blocker. Although a wide variety of biomembranefusion agents and potassium channel blockers that are mentioned abovemay be included in the composition, a preferred biomembrane fusion agentis a polyalkylene glycol and a preferred potassium channel blocker is asubstituted pyridine. In more preferred forms of the invention, thepolyalkylene glycol is polyethylene glycol and the potassium channelblocker is an amino-substituted pyridine, such as 4-aminopyridine. Thecomposition may be in a pharmaceutically acceptable carrier as describedabove.

Although the methods and compositions of the invention are useful intreating a wide variety of vertebrates, they may be advantageously usedto treat mammals and preferably humans. Moreover, although the methodsand compositions are advantageously and surprisingly useful in treatingthe spinal cord, they may also be used in treating the peripheralnervous system and/or central nervous system, or other areas in whichdamaged axons are present.

Reference will now be made to specific examples illustrating thecompositions and methods described above. It is to be understood thatthe examples are provided to illustrate preferred embodiments and thatno limitation to the scope of the invention is intended thereby.

EXAMPLE 1 Acute In Vitro Response of Crushed Spinal Cord to PEG

This example demonstrates that compound. action potentials are restoredin a compressed spinal cord in vitro after it is treated with PEG.

In Vitro Isolation of the Spinal Cord

Adult female guinea pigs of 350-500 gram body weight were used for thesestudies. The spinal cord was isolated from deeply anesthetized animals[(60 mg/kg ketamine hydrochloride, 0.6 mg/kg aoepromazine maleate, and10 mg/kg xylazine, intramuscularly (i.m.)]. Following anesthesia, theanimal was perfused transcardially with cold (150C) Krebs' solution(NaCl, 124 mM; KCl, 2 mM; KH2PO4, 1.2 mM; MgSO4, 1.3 mM; CaCl₂, 11.2 mM;dextrose, 10 mM; NaHCO3, 26 mM; sodium ascorbate, 10 mM; equilibratedwith 95% O₂, and 5% CO₂). The vertebral column was rapidly removed usingbone forceps and scissors by previously described techniques [Shi, R.and Blight, A. R. (1996) J. of Neurophysiblogy, 76(3): 1572-1579; Shi,R. and Blight, A. R. (1997) Neuroscience 77(2): 553562]. The spinal cordwas divided into four longitudinal strips, first by midline sagittaldivision, then by separating the dorsal and ventral halves with ascalpel blade against a plastic block. Only the ventral white matter wasused for this study. These 35-38 mm long strips of spinal cord whitematter will usually be referred to below as “cords” or “spinal cords”for ease of description. Spinal cords were maintained in continuouslyoxygenated Krebs' solution for an hour before mounting them within therecording chamber. This was to ensure their recovery from dissectionbefore experiments were begun.

Double Sucrose Gap Recording Technique

The double sucrose gap recording chamber is shown in FIGS. 1A and 1B andhas already been described in previous publications [Shi, R. and Blight,A. R. (1996) J. of Neurophysiology, 76(3): 1572-1579; Shi, R. andBlight, A. R. (1997) Neuroscience 77(2): 553-562]. Briefly, the strip ofisolated spinal cord white matter was supported in the three-compartmentchamber. The central compartment was continuously superfused withoxygenated Krebs' solution (about 2 ml/min) with a peristaltic pump. Thecompartments at both ends were filled with isotonic (1120 mM) potassiumchloride, and the gap channels with 230 mM sucrose. The white matterstrip was sealed on either side of the sucrose gap channels with shapedfragments of glass coverslips that also blocked the flow of fluid in thenarrow gap between the coverslip and the tissue surface. Note that thecentral chamber is at ground potential for recording. The sucrosesolution was run continuously through the gap at a rate of 1 ml/min.Axons within the spinal cord strip were stimulated and compound actionpotentials (CAPs) were recorded at the opposite end of the white matterstrip by silver-silver chloride electrodes positioned within the sidechambers and the central bath as shown in FIG. 1B. Specifically, actionpotentials were stimulated at the left side of the spinal cord strip asshown in the figure, conducted through the spinal cord in the centralcompartment (also including the injury site), and recorded at the rightside of the spinal cord strip as shown. Stimuli were delivered throughstimulus isolation units in the form of 0.1 msec constant currentunipolar pulses. A conventional bridge amplifier with capacitycompensation (Neurodata Instruments) was used to amplify the signal.This data was digitized and stored on video tape with a NeurodataInstruments Neurocorder for subsequent analysis. During the experiment,the oxygenated Krebs' solution continuously perfused the isolated spinalcord tract, while temperature was maintained at 37° C.

Every electrophysiological test was digitized in real time and capturedto the computer for subsequent quantitative evaluation. All records werealso recorded on VHS magnetic tape as a means of back up documentation.All solutions used in the PEG repair process were made on the day oftheir use.

The Compression Injury

A standardized compression injury was produced with a stepper-motorcontrolled rod which compressed the spinal cord while suspended insidethe recording chamber (FIG. 1B). Briefly, the isolated white matterstrip was compressed against a flat, raised plastic, plexiglass stage atthe center of the recording chamber with the flattened tip of aplexiglass rod. The tip was advanced downward to contact the tissue at astandardized rate of about 25 pm/s. The downward movement of the rod wascontrolled with a stepper motor to produce a finely graded crush justsufficient to eliminate all CAP propagation (which was monitoredcontinuously during the procedure). The end of the rod with theflattened tip provided a compression surface of 2.5 mm along the lengthof the tissue, and a transverse width of 7 mm, such that it was alwayswider than the spinal cord strip, even under full compression.Positioning of the compression rod was accomplished with amicromanipulator. CAPs were simultaneously recorded during the injuryprocess. Compression was stopped when CAPs were completely eliminated.The state of complete CAP failure was maintained for an additional 15seconds before the rod was rapidly withdrawn from the cord's surface torelieve pressure. The recovery of the CAP was then documented. The basicrecovery profile following such standardized compression in normalKrebs' solution has been previously characterized and published [Shi, R.and Blight, A. R. (1996) J. of Neurophysiology, 76(3): 1572-1579]

PEG Repair Procedure

The PEG repair procedure included the following steps:

1) Typical physiological functioning of the isolated white matter stripremoved to the recording chamber required about ½ to 1 hour ofincubation time while immersed in oxygenated Krebs' soluction tostabilize. In initial experiments, once the CAP propagation hadstabilized, the Krebs' solution was replaced with Ca²⁺-free Krebs' (Ca²⁺replaced with an equimolar amount of Mg²+).

2) The spinal cord strip was then crushed by the techniques describedabove, while simultaneous stimulation and recording continued.

3) A solution of PEG in distilled water (50% by weight) was applied by apressure injection through a micropipette. A vital dye was added to thePEG solution to monitor its continuous application to the lesion site ina stream about 0.5 mm wide for about 1-2 minutes. The PEG was applied toone side of the lesion, washed over it, and immediately removed byconstant aspiration on the other side using a second pipette.

4) Immediately following the PEG application, the bathing media in thecentral chamber was replaced with a continuous stream of oxygenatednormal Krebs' solution. The physiological properties of the PEG-treatedspinal cord were monitored continuously for 1 hour. Usually, a weakrecovering CAP was evident within 6-15 minutes of the PEG application.

The above-described technique should be considered as a basic one, fromwhich testing of several variations described below was performed. Forexample, tests were made of the response of “recovering” axons to theadditional application of the fast potassium channel blocker, 4aminopyridine (4-AP). In this trial, 5 separate cords were treated withan application of PEG as described above and compared to 5 controlcords. One hour after compression, 100 pM 4-AP (in Krebs' solution) wasapplied for 15 minutes and then washed free with normal Krebs's olutionas described above.

In a final series of experiments, a determination of whether it wasnecessary to carry out the methods of the present invention in Ca²⁺-freemedia was made. In these experiments, the cord was compressed while Rwas immersed in normal Krebs' solution.

Statistical Treatment

Before and after the application of 4-AP, Student t tests were used 20to compare recovering action potential, amplitude between the controland PEG-treated group. Comparisons of action potential amplitude werealso made between the two PEG-treated groups.

Results

PEG-Mediated Repair of Crushed Spinal Cord Strips

Approximately {fraction (1/2)} hour following the equilibration of thespinal cord strip in the recording chamber, the Krebs' solution in thecentral compartment was replaced with a Ca²⁺-free Krebs' and the spinalcord was crushed by previously described techniques. In every spinalcord tested in this group of twenty (ten control and ten experimental),this procedure resulted in the immediate and total loss of CAPpropagation from the point of stimulation to the point of recording.FIG. 2 shows an individual record of one typical control experiment anda PEG-treated experimental spinal cord strip. Note the immediate andcomplete loss of the CAP in both preparations, and the initial recoveryof the CAP in the PEG-treated spinal cord by 5 minutes post treatment(FIG. 2, lower panel). Note that at the earliest time point (about 5minutes post injury) seen in FIG. 2, recovery of a CAP is never observedin the absence of PEG treatment and rarely occurs by 10 minutes postinjury (latter data not shown). The earliest recorded recoveries of aCAP occurred within 1-2 minutes following PEG treatment. In controlpreparations, 3 cords never regained conduction during the 1 hour ofcontinuous observation. In contrast, not one PEG-treated spinal cordproviding the data summarized in FIG. 3 failed to recover CAP conductionfollowing PEG treatment. In four more control spinal cords, the recoveryof the CAP was not observed for approximately twenty minutes.

FIG. 3 provides a summary graph of the 10 control and the 10experimental spinal cords treated and monitored identically, except forthe experimental application of PEG to the lesion site. Note that thecontrol group shows a barely detectable CAP (3.6%) even by 1 hour postinjury, while average recovered CAPs in PEG-treated cords increaseapproximately −19%, ranging to as much as 69% of the pre-crushamplitude. PEG treatment always (1) provided a striking increase in theamplitude of recorded CAPs, averaging 19% of the original pretransectionamplitude and (2) facilitated the CAP recovery in 100% of the casestested. At every time point tested, including the 10 minute post injuryperiod, recovered CAP amplitudes were statistically significantlygreater than control preparations (P<0.05, Student's t test,two-tailed). CAP recovery was facilitated when the injury was notcarried out in Ca²⁺-free Krebs'solution. The amplitude of the recoveredCAP in normal Krebs' at the first time point (10 minutes post injury)was statistically elevated over the recovered CAP observed when theinjury was performed in Ca²⁺-free media (P<0.05; unpaired Student ttest). Every subsequent time point was still higher in this data setwith no reverse trends, but without statistical significance. Thus, itis seen in FIG. 3 that the injury need not be carried out in Ca²⁺-freemedia to produce functional repair as claimed by Bittner forinvertebrate axons [Krause, T. L. and Bittner, G. D. (1990) PNAS 87:1471-1475].

Electrophysiological Properties of the Repaired Spinal Cords

The PEG-repaired spinal cords showed typical conduction properties (asobserved in recovering untreated cords) however some differences intheir electrophysiological properties were revealed by furtherevaluation.

FIG. 4A shows the effect of injury on the normal recovery of CAPamplitudes. Typically, the recovered CAP was dampened in amplitudeacross all threshold intensities of excitation. It was also evaluated ifthis reduced magnitude of the CAP occurred across all caliber spectra ofinjured axons within the spinal cord strip, or was manifest in onlylarge or small diameter axons. FIG. 4B shows the actual amplitudes ofcontrol compound potentials at 1 hour post injury, plotted against thepreinjury amplitude at the same stimulus intensity. A less severe injurywas required in these spinal cords to allow an adequate range ofrecovered CAP amplitudes, for this graded evaluation. In the severelyinjured cords, the maximal recovered CAPs were insufficient toadequately make these comparisons. These data points are shown relativeto the maximum amplitude achieved prior to and after injury. A leastsquares linear regression was not significantly different from 1:1linearity, suggesting that there was no difference between thesusceptibility to damage of axons of different stimulus thresholds.

In FIG. 4C, two hypothetical lines are plotted, representing outcomesfollowing PEG treatment. Note that if larger axons of a lowered stimulusthreshold were more susceptible to PEG, the data would be shifted as inline (a). In the opposite situation, the hatched line (c) shows a shiftin the opposite direction should small caliber axons with a higherstimulus threshold be repaired. In FIG. 4D, the actual data taken fromthe PEGtreated population is plotted in the same manner as in FIG. 4B.Note that the least square linear regression line is not significantlydifferent from 1:1 linearity, which is again not different from thatshown in FIG. 4B. The near unity slope of the relation of amplituderesponse before and after injury indicated no consistent selectivity ofPEG-mediated improvement of conduction in fibers of lower or higherthreshold. In this test, the typical and severe standardized injury wasused, since PEG-repaired cords showed substantial CAPs sufficient for agraded plot of their amplitudes.

Although PEG appeared to be able to repair axons of a wide range ofcalibers similar to the natural recovery process observed in controlcords, the electrophysiological properties of PEG-mediated recoverieswas not the same as controls. FIG. 5A shows the classical relationshipbetween the timing of paired stimuli and the amplitude of the twoelicited CAPs. Paired stimuli in which the interstimulus interval wasbetween 0.6 to 15.0 ms demonstrated typical dampening of the CAPamplitude soon after the absolute refractory period. When the intervalbetween the paired stimuli was longer than this, a plateau was reachedwhere the first and second CAPs were of an identical magnitude, markingthe extent of the relative refractory period.

FIG. 5B shows control data derived from 4 separate experiments. Theabscissa shows the magnitude of the second CAP of the pair as a percentof the magnitude of the first elicited CAP. The ordinate shows the logof the interstimulus interval ranging from 0.6-15 ms. This sigmoidalplot is typical, beginning with stimuli that do not elicit a second APduring the absolute refractory period, and ending at the termination ofthe relative refractory period.

Furthermore, FIG. 5B shows that this relationship was not disturbed bythe injury, as pre- and postinjury data points were not significantlydifferent along this sigmoidal curve. This did not hold true, however,for PEG-treated spinal cords. The early and robust recovery of CAPsproduced by PEG demonstrated a typical period of absolute refractory asbefore the injury and experimental treatment. Furthermore, the relativerefractory period also appeared to terminate when a similar stimulusinterval to control preparations was achieved. During the refractoryperiod of PEG-treated cords, the amplitude of the second CAP wasslightly reduced when compared to that before the crush and PEGtreatment (FIG. 5C). However, this latter relationship was notstatistically significant.

EXAMPLE 2 Potassium Channel Blockade as an Adjunct to PEG-MediatedRecovery of Conduction

This example shows that treatment of injured spinal cords in vitro withboth a potassium channel blocker and a biomembrane fusion agent allowssynergistic recovery of CAPs.

It is a common feature of injured cells to loose intracellular potassiumto the extracellular milieu through compromised membrane. In axons, thismay be sufficient to suppress action potential conduction. Thus, it wasattempted to determine if blockage of fast potassium channels with 4-APwould affect the properties of conduction immediately following PEGrepair.

Spinal cords were crushed, isolated and treated with PEG as described inExample 1. Analysis was also performed in the double sucrose recordingchamber as described in Example 1.

FIG. 6A shows the enhancement of the CAP in crushed (but untreated withPEG) spinal cord by 4-AP. In this individual record, the initialrecovered CAP at 1 hour post injury is shown, and the enhanced CAPfollowing 100 pM 4-AP treatment is superimposed upon it. Followingdocumentation of the 4-AP enhanced CAP, the blocker was washed out, andthe media in the central compartment was replaced with normal Krebs'solution. The CAP fell to pretreatment levels by 15 minutes and wasindistinguishable from the original record. This final waveform issuperimposed on the other two CAPs in FIG. 6A but cannot bediscriminated from the pretreatment electrical record. In this singletest, 4AP reversibly enhanced the recovered CAP by about 40%.

FIG. 6B shows an identical test performed on a PEG-treated spinal cord,in which 4-AP was administered at 1 hour post PEG application. In thisindividual test, the second CAP was reversibly enhanced by about 70%.

Following the near doubling of the CAP, 4-AP was washed out asdescribed, and the CAP fell to pretreatment levels as in controls (FIG.6A).

FIG. 6C shows the group data, including 5 spinal cords in each group.The percent enhancement of the PEG-mediated recovery for the group datamirrors that discussed above for the individual experiments (about 70%enhancement in the experimental group; about 40% in the control group).This experimental enhancement was statistically significantly greaterthan that observed in the controls. (p<0.05, unpaired Student's t test)

Although not being limited by theory, FIG. 7 depicts a proposedmechanism of the synergistic effect of PEG and 4-AP. A severe mechanicalcompression of a myelinated axon is diagrammed at the top. Note that themyelin sheath envelops high densities of fast W channels clustered atthe paranodal region. Severe crush leads to an exposure of the potassiumchannels of the paranodal region by a withdrawal or collapse of themyelin lamella at this site [Shi, R. and Blight, A. R. (1996):Neuroscience, 77: 553-562]. Exposure of the voltage gated potassiumchannels after injury would elevate K⁺ conductance further impedingconduction across this damaged portion of the membrane (gray regionshowing “holes” in the compromised membrane). In control preparations,partial to complete conduction block results from this localizeddisturbance of the axolemma, which may progress to complete separationof the axon and loss of the distal axonal segment by Walleriandegeneration (left side of FIG. 7). In PEG-treated axons (right side ofFIG. 7), the membrane repair leads to preservation of injured axons aswell as improvements in their conduction capabilities (gray regions;membrane holes now sealed). However, elevated K⁺ conductance through K⁺channels that are still exposed at the site of repair in PEG-treatednerve fibers might still suppress conduction to some extent. Blockade ofthese channels with 4 AP (FIG. 7, small arrow heads; lower right) wouldbe expected to reduce any outward K⁺ conductance and thus enhanceconduction.

SUMMARY OF RESULTS

Within a few minutes after the application of the water-soluble polymerPEG, an immediate recovery of CAP propagation through the lesionoccurred. The recovered CAP amplitude slowly increased with time to apeak of about 20% of the initial CAP amplitude. Moreover, this level ofrecovery a) was always statistically significantly higher than controlamplitudes, b) was observed at every time point tested, and c) occurredin 100% of the experimentally treated spinal cords. It is clear that atopical application of PEG can immediately repair severe compressioninjury to the mammalian spinal cord leading to significant increases infunctional recovery as defined by the enhanced capacity to propagatenerve impulses through the lesion. This report is the first todemonstrate PEG-mediated repair of crushed mammalian nervous tissue.

We have shown that a physiological, balanced media and theaforementioned PEG solution, is all that is required to producefunctionally significant repair in mammalian spinal cords (see below).Moreover, in other experiments, where completely transected guinea pigspinal cords were fused with PEG, it has been revealed there was nospecific PEG molecular weight critical to the process, having tested PEGsolutions using 400, 1400, 1800, 2000, and 3700 daltons (unpublishedobservations).

In this physiological study, similarities and differences between thenatural mechanisms of axonal repair and those mediated by PEG have beendetermined. First, a least squares linear regression analysis of preandpostinjury CAP amplitudes suggests that PEG-mediated repair can occuracross all levels of stimulus thresholds, reflecting axon diameters, asdoes the natural recovery process in untreated spinal cord strips. Inother words, all spinal axons regardless of their caliber are equallysusceptible to PEG mediated repair [see Shi, R. and Blight, A. R. (1996)Neuroscience 77: 553-562 for a similar analysis of axonal recovery fromcompression injury]. The differences between natural repair and thatproduced by PEG application are more striking. First, this injury isvery severe; 30% of control spinal cords never recovered any capacity toconduct CAPs during the 1 hour period of evaluation following injury. Onthe other hand, there was no instance where PEG did not initiate ameasurable physiological recovery. On a more subtle level, there appearsto be a slightly reduced CAP amplitude during the period of relativerefractory in only PEG-mediated CAPs relative to control cords. Oneexplanation for this observation may be that in control cords a severelycompromised and dysfunctional population of axons may become completelynonfunctional, revealing more normal conduction properties in thatpopulation that survive the injury. PEG may rescue a portion of suchseverely compromised axons, recruiting them into the CAP, and perhapsaccounting for its slightly different conduction properties.

The above-described in vitro evaluation of the anatomy of axonal repairfollowing mechanical compression has revealed that a 2 minuteapplication of PEG produced sealing of membrane lesions at the site of astandardized compression. Sealing was indicated by the exclusion ofhorseradish peroxidase uptake by injured fibers in the PEG-treated groupcompared to sham-treated spinal cords (J. Neurocytology, 2001, inpress). Such immediate repair of membrane breaches sufficient to inhibitthe uptake of large molecular weight dyes should also arrest or reducepermeabilization, allowing the nonspecific flux of ions across it.Although not being limited by theory, it is believed that this “sealing”behavior of PEG both restores excitability and reverses anatomicaldissolution of the nerve fiber.

This procedure may advantageously applied to treat severe, acuteneurotrauma. In addition to immediate improvements in conduction, repairof crushed axons in peripheral nerves leading to a rescue of theirdistal segments would provide the added benefit of reducing atrophy ordegeneration of target cells or so called “end organs.” Moreover,PEG-mediated fusion of even transected axons could become a component ofmicrosurgical grafting techniques since the conventional resection ofperipheral nerve trunks prior to fasicular grafting exposes the severedtips of proximal and distal axonal segments, making them available forfusion.

EXAMPLE 3 Effect of PEG on Restoration of CAPs In Severed Spinal CordAxons

This example demonstrates that severed spinal cord axons may be fusedwith PEG, thus allowing restored conduction of CAPs through the lesionsite.

In Vitro Isolation of Spinal Cord

The spinal cord of adult female guineas pigs was isolated according tothe protocol of Example 1. After the cord was isolated, it was halved bymidline sagittal division. The ventral white matter was separated fromgray matter with a scalpel blade against a soft plastic block. Cordswere maintained in continuously oxygenated Krebs' solution for at leastan hour before mounting in the recording chamber. This was to ensure therecovery from dissection before each experiment was begun.

Double Sucrose Gap Recording Technique

The technique was followed according to the protocol in Example 1. Thecentral bath was connected to instrument ground. The entire chamber wasmounted on a Peltier temperature control system, which also maintainedthe entire preparation at 370C. Thermistors, in the chamber next to thespinal cord, constantly recorded and displayed the temperature. Aftermounting the spinal cord in the sucrose gap chamber, recorded CAPs andcompound membrane (Gap) potentials usually stabilized with an hour [Shi,R. and Blight, A. R. (1996) J. of Neurophysiology, 76(3): 1572-1579;Shi, R. and Blight, A. R. (1997) Neuroscience 77(2): 553-562]; Shi, R.and Borgens, R. B. (1999) J. Neurophysiblogy, 81: 2406-2414.

PEG Fusion Procedure

The basic methodology used to fuse spinal axons was as follows:

1). Restoration of typical physiological functioning of the isolatedwhite matter strip removed to the recording chamber required about ½ to1 hour of incubation time while immersed in oxygenated Krebs' at 37° C.Once both the Gap potential and CAP propagation were normal, the spinalcord strip was transected.

2). The spinal cord strip was completely severed with a laboratoryfabricated cutter (a shard of a razor blade attached to an applicatorstick), and the two ends of the spinal cord were observed to beseparated by a gap of about 0.5-1 mm with a stereomicroscope. The spinalcord was transected within the middle of the central compartment of therecording chamber. Stimulation and recording were continued during thisoperation. Following transection, the two ends of the cord segments were“pushed together,” i.e., abutted tightly using a watchmaker forceps anda laboratory fabricated device that applied gentle pressure on onesegment of the spinal cord strip pressing and holding it against theother. The device was mounted on a micropositioner, and contacted thespinal cord parenchyma with a strip of nylon mesh stretched across twometal bands (FIG. 8). The metal frame of the device never contacted thespinal cord tissues during use. Only the nylon mesh was in contact withthe tissue. Several methods to accomplish stabilization during thefusion process were tested, the most effective involved first lightlyplacing the mesh onto the intact cord. Once this was accomplished, thespinal cord strip was completely transected, with a gap appearingbetween the two segments which were then repositioned as discussedabove.

3). Various solutions of PEG (1400, 1800, 2000, and 3500 daltons, 50% byweight in distilled water) were applied by pressure injection through amicropipette in preliminary experiments (data not shown), while the datareported here exclusively utilized PEG of having a molecular weight ofabout 1800 daltons. A vital dye was added to the PEG solution to monitorits application to the lesion site as a continuous stream about 0.5 mmwide and continuing for about 1-2 minutes. The PEG was applied to oneside of the lesion, washed transversely across it, and was removed byconstant aspiration on the other side using a second suction pipette.

4). During the PEG application, a continuous stream of oxygenated Krebs'solution was maintained. The electrophysiological properties of thespinal cord following PEG treatment was monitored continuously forapproximately 1 hour.

Results

Typical CAPS were recorded in response to stimulation, and werecompletely eliminated following transection of the cord between thestimulation and recording electrodes in every spinal cord strip tested(FIGS. 9A and 9B). The recovery of CAPs was often observed within 5-15minutes following PEG application and continued for up to 60-80 minutes,at which time physiological recordings were discontinued (FIGS. 9C and91). Since the conduction of CAPs across the plane of transection doesnot occur in severed spinal cords, a fusion was defined as successful ifa restored CAP was detected demonstrating properties of latency andstimulus threshold. In preliminary experiments, it was discovered thatthe success of an attempted fusion depended on the alignment and thecare taken during abutment of the spinal cord segments prior to PEGapplication. The ends of the strips cannot be too tightly forcedtogether or this produces more injury to the spinal cord. They cannot betoo loosely abutted or fusion of the axolemmas will not take place (seebelow). All 20 of the attempted fusions reported here were succesful.Recovered CAPs were on the order of 5% of the peak magnitude of theoriginal pre-transection CAPs. Note that the computer managed dataacquisition techniques used in to obtain physiological recordings shownin FIGS. 9A-9I have been previously reported [Shi, R. and Blight, A. R.(1996) J. of Neur7ophysiblogy, 76(3): 15721579; Shi, R. and Blight, A.R. (1997) Neuroscience 77(2): 553-562]

Table 1 below provides the quantitative data derived from an evaluationof 20 successful fusions using 1800 dalton PEG. In preliminaryexperiments, identical, functional fusions were achieved in a few casesusing 1400, 2000, and 3500 dalton PEG (data not shown). TABLE 1Characteristics of 20 successful fusion of mammalian spinal cord axonsutilizing 1800 dalton PEG. AP amplitude Peak latency (ms) ½ HeightDuration (ms) Exp#¹ Pre² post³ (%)⁴ SEM⁵ range⁶ pre post (%) SEM rangepre post (%) SEM range 20 2.44 0.44 4.61 2.83 0.2-58.0 0.93 1.05 114.58.71 26-227 0.63 0.53 85.5 6.45 36-156¹Total number of fusions carried out at 370 C.²Data obtained before transection.³Data obtained after PEG-mediated fusion.⁴Mean percent recovery after fusion⁵Mean standard error after fusion.⁶Range of data.

A series of control procedures were performed to insure that restoredCAP conduction was indeed a function of restored axonal integrity andnot produced as an artifact by some alternate means of conduction. Forexample, CAPs were not conducted across the plane of transection if: (1)subthreshold stimulation was applied to PEG fused cords (20 cases), (2)the original fusion site was again transected with the cutting device (6cases, FIG. 9D), (3) spinal cord segments were closely abutted in thestream of Krebs's solution, but PEG was not applied (5 cases, FIGS. 9Eand 9F), and (4) PEG was applied to poorly abutted segments (5 cases,FIGS. 9G and 9H).

EXAMPLE 4 Effect of PEG on Anatomical Continuity of Severed Axons

This example illustrates that PEG fuses and repairs severed axons suchthat intracellular fluorescent dyes may diffuse across the originaltransection. Moreover, the restored anatomical continuity is shown to becorrelated with the restored ability to conduct CAPs.

Tract Tracing with Intracellular Fluorescent Probes

Intracellular injections of two fluorescently decorated dextrans wereused to evaluate the integrity of formerly transected nerve fibers byprocedures previously described [Borgens, R. B. and Bohnert, D. M.(1997) Exp. Neurol. 145: 376-389]. Briefly, injections of about. 1-1.5μl of one tracer, tetramethylrhodamine dextran or Flouroruby (FIR, 8000dalton, Molecular Probes Inc.), was made to one segment of the fusedcord, approximately 4-6 mm from the original plane of transection. Thislabel was observed with excitation/barrier wavelengths of 545/590 nmrespectively, in darkfield. Likewise, a second and similar injection ofanother tracer, FITC conjugated dextran or Flouroemerald (FE, 8000dalton, Molecular Probes, Inc.) was made to the opposite segment andobserved with excitation/barrier wavelengths of 495/515 nm,respectively.

Approximately 12-14 hours later the cords were immersion fixed in 5%glutaraldehyde/0.01% paraformaldehyde. This time period allowed theintracellular markers to diffuse throughout axons. During thisincubation period, a continuous flow of oxygenated Krebs' solution wasmaintained through the central compartment of the recording chamber,which helped to eliminate any extracellular diffusion of the dye.Longitudinal horizontal sections (about 15-30 μm) of the spinal cordstrips were made on either a freezing microtome or the tissue wasimbedded in paraffin for sectioning by conventional histologicaltechnique. Each of these dyes was viewed independently using theappropriate barrier and filter combinations in fluorescent darkfield.These operations were performed on 8 PEG-treated, and fused spinal cordstrips. Additional control procedures were performed on 5 spinal cordstrips to insure that dye did not travel into the opposite segment ofthe cord from where it was injected. This involved injecting the twodyes into cord segments as previously described; however, these segmentswere tightly abutted but not fused with PEG.

In another 5 PEG-treated spinal cord strips, high resolution lightmicroscopy was used to evaluate the plane of fusion. These fixed stripsof spinal cord were cut to a length of about 5 mm containing the fusionplane, embedded in plastic by conventional methods, sectioned at 0.5-1micron on an ultramicrotome, and stained with 1% toluidine blue.Microscopic images were captured directly to a Dual Pentium PRO PC froman Olympus Van Ox Universal microscope fitted with an Optronics DEI-7503 CCD video camera system.

Results

The anatomical continuity of axons that had been fused at the plane oftransection was correlated with the restored ability to conduct CAPs in13 spinal cords. This was determined by the intracellular diffusion ofthe two different fluorescent dyes across the lesion site in 8 cords andin 5 additional cords by conventional microscopy of plastic imbeddedsections. Axons filled with the different dyes were examinedindependently of each other by fluorescence microscopy with differentexcitation and barrier filter combinations in darkfield. FIGS. 10A-10Dare typical of all 5 control preparations (cord segments tightly abuttedbut without PEG application) in which the potential of the dye todiffuse from one segment to the other by an extracellular pathway wasexamined. In all controls, a dye injected into axons of one segment wasnever observed within axons of the opposite cord segment. This was inpart due to the small dye volume injected (about 1.5 μl) coupled to acontinuous flow of media through the central compartment of therecording chamber maintained during the entire procedure. Thiseliminated the spread of dye by an extracellular diffusion path.

In both control and experimentally fused cords, unfused axons retractedback from the transection plane for a variable distance (50 μm-0.5 mm),their terminal clubs or endbulbs clearly visible (FIG. 11C). The planeof transection was identifiable in PEG-fused cords as a transverseseries of gaps and holes interspersed with well-fused regions of spinalcord parenchyma (FIGS. 11A, and 11D-11F). It is believed that such gapsresult from partial separation of the fused segments during handlingprior to fixation as well as incomplete perfusion of the cord with PEG.Since PEG probably fuses some non-neuronal cells as well as neuronalprocesses, the original transection plane in well fused expanses ofspinal cord was nearly undetectable with fluorescent microscopysufficient for visualizing the two intracellular dyes. However,blue-violet fluorescent illumination induced some spinal cord autofluorescence which could reveal the fusion plane in these regions as avery fine “seam”.

In all 8 cords in which tract tracing was performed, a recovered CAP wasdocumented. In 6 of these, fibers filled from either end of the cordtraversed the fusion plane into the adjacent segment. In 2 of these 6strips, only fibers filled with FR crossed the lesion, the FE labeledsegments being poorly filled. It was common to observe the terminal endsof unfused axons within a few micrometers of fused axons adjacent tothem which filled along their lengths across the original transection(FIG. 11C). In one case, axons were observed to have fused to two ormore others, producing a tangle of nerve fibers within the transectiongap (FIGS. 11A and 11B). This tangle of fibers could also be traced tothe opposite spinal cord segment. Additionally, note that theapproximate site of dye injection in FIG. 11A is to the left of theimage and out of the field of view. In three of the five cords evaluatedby high resolution light microscopy, unmyelinated expanses of myelinatedaxons fused at the original plane of transection (FIGS. 11E and 11F). Inthese regions membrane and myelin debris could also be seen in the gapsurrounding the reattached fibers. Furthermore, in FIG. 11E, the two,re-apposed ends of the white matter strip show continuity over a lengthof apposition in the middle of the frame. The plane of transection(dashed line) is clear from the slight gaps that remain between the endsof the strip at the top and bottom of the figure, which continue acrossthe whole width of the tissue in the rest of the section (visible atlower magnification).

SUMMARY OF THE RESULTS

Our data show that a water soluble polymer, PEG, can be used to rapidlyreconnect the severed halves of spinal axons within completely severedstrips of isolated spinal cord white matter. This fusion has beendocumented by both anatomical and physiological means. In the former,fluorescent intracellular markers were injected into each spinal cordsegment. In 6 cases of 8, axonal continuity across the plane of fusionwas demonstrated. By searching the fusion plane in plastic embeddedsections, unambiguous evidence of axonal fusion in three of fiveadditional spinal cord strips was detected.

In all five control spinal cords, the intracellular label was neverobserved in the opposite cord segment from where it was injected. Thiswas largely due to the elimination of an extracellular diffusion pathwayfrom the site of injection to the opposite cord segment by the flowingmedium in the central compartment and the separation of compartments bythe sucrose gap “boundaries”. Furthermore, the presence of numerousterminal clubs of transected but unfused axons adjacent to well-labeledaxons crossing the transection plane provided additional anatomicalevidence that true fusion of severed proximal and distal axon segmentshad taken place.

The immediate restoration of CAP propagation across the transectionplane in completely severed spinal cords following PEG treatment couldonly have occurred coincident with the functional reconnection ofproximal and distal segments of axons. In control spinal cords, CAPconduction did not reappear following tight abutment of the severedsegments when PEG was not applied. Furthermore, CAP conduction did notreappear in segments that were poorly abutted by uesign and also treatedwith PEG. Thus, PEG itself does not provide some sort of substratepermitting CAP conduction. It is to be concluded that a topicalapplication of PEG indeed functionally reunites severed mammalian nerveprocesses. This observation compliments and extends the demonstrationthat topically applied PEG can repair guinea pig spinal cords severelycrushed by a standardized procedure leading to an immediate recovery ofaction potential propagation through the lesion [Shi, R. and Borgens, R.B. (1999) J. Neurophysiol. 81: 2406-2414].

EXAMPLE 5 In Vivo Effect of PEG on Restoration of the CTM Reflex inGuinea Pigs with Crushed Spinal Cords

This example illustrates that in vivo treatment of crushed guinea pigspinal cords restores the CTM reflex.

Surgery and Anesthesia

A total of 51 adult (300 gm) guinea pigs were used in two separateexperiments. Guinea pigs were anesthetized with an intramuscularinjection of 100 mg/kg ketamine HCL, and 20 mg/kg xylazine, and thespinal cord was exposed by dorsal laminectomy [Borgens, R. B., et al.(1986) J. Comp. Neurol. 250: 157-167; Borgens, R. B., et al. (1990) J.Comp. Neurol. 296: 634-653]. Subsequently, a constant displacement 15second compression of the spinal cord was performed using a modifiedforceps possessing a detente [Blight, A. R. (1991) J. Neurolog. Sci.103: 156-171]. In this experiment, the lesioning procedure hadpreviously been calibrated to produce an immediate and total loss of CAPconduction through the injury and behavioral functioning of thecutaneous trunci muscle reflex (see below). For some SSEP measurements,or to sedate animals for behavioral testing and videotaping, guinea pigswere injected with 0.1 cc Na⁺ Pentobarbital, 50 mg/ml. Surgery andfunctional testing were carried out under protocols approved by thePurdue University Animal Care and Use Committee, in accordance withFederal, State, and University guidelines governing animal use inresearch.

PEG Application

An aqueous solution of PEG (either 400 or 1800 daltons, 50% by weight indistilled water) was applied with a pipette to the exposed injury fortwo minutes in experimental animals, and then removed by aspiration. Asin prior in vitro experiments [Shi, R. et al. (1999) J. of Neurotrauma16: 7277 38; Shi, R. and Borgens, R. B. (1999) J. Neurophysiology 81:2406-2414], no difference in the response to these two solutions wasdetected, so these data are pooled in this report. The site of PEGapplication was immediately lavaged with isotonic Krebs' solution (124mM NaCl, 2 mM KCI, 1.24 mM KH₂PO₄, 1.3 mM MgSO₄, 1.2 mM CaCl₂, 10 mMdextrose, 26 mM NaHCO₃, and 10 mM sodium ascorbate), and any excess PEGand/or Krebs' solution removed by aspiration. Although PEG was notapplied to the injury in sham-treated animals, the site was lavaged withKrebs' solution which was subsequently removed by aspiration. The woundswere closed, and animals kept warm until awaking with heat lamps. Guineapigs were housed individually and fed ad libidum.

In the first experiment, it was attempted to repeat the remarkablecomplete reversal of functional loss within minutes of severe spinalinjury as observed in in vitro trials [Shi, R. et al. (1999) J. ofNeurotrauma 16: 727-738; Shi, R. and Borgens, R. B. (1999) J.Neurophysiology 81: 2406-2414]. Thus, PEG was applied within about 15minutes of spinal cord compression (experimental n=14, control n=11). Inthe second experiment, PEG application was delayed for about 8 hours(experimental n=11, control n=11). The former groups were evaluated forabout 4 days, and the latter, for about 1 month, after PEG application.In both experiments, documentation of CTM behavior was combined withphysiological recording.

An additional 4 PEG-treated animals were followed for 1 day post injuryat which time their spinal cord was again exposed at the site of theoriginal injury and crushed again at this location using the sametechnique as reported above.

Behavioral Analysis of the Cutaneus Trunci Muscle (CTM) Reflex

The CTM behavior is observed as a corrugated rippling of backskin inresponse to light tactile stimulation (FIG. 12B). The behavior isdependent on afferent sensory projections organized as a long tract ofaxons in each ventral funiculus of the spinal cord, just lateral to thespinothalamic tract [Blight, A. R., et al. (1990) J. Comp. Neurol. 296:614633; Thierault, E. and Diamond, J. (1988) J. Neurophys. 60: 446-463;Thierault, E. and Diamond, J. (1988) J. Neurophys. 60: 463-477].

Specifically, FIG. 12A shows a diagram of the sensory and motorcomponents of the CTM reflex of the guinea pig. Sensory receptors inbackskin project afferent axons into each thoracic segment on both sidesvia the dorsal cutaneous nerves (dcn). These enter the spinal cord andsynapse on second and third order neurons which project their axons(red) to the thoracocervical junction. These tracts of ascending nervefibers are located on each side of the spinal cord within the ventralfunniculus, lateral to the spinothalalmic tract. These ascending axonssynapse on bilaterally located pools of CTM motor neurons locatedbetween T-1 and C-6. Motor fibers exit the spinal cord on each side as acomponent of the brachial plexus and innervate the cutaneous truncimuscle of the skin. Note that a spinal cord lesion extending across bothsides of the cord compromises ascending tracts, producing a region ofbackskin areflexia on both sides below the level of the injury. In thisregion of skin, tactile stimulation no longer elicits skin rippling.

The reflex is bilaterally organized as segmental receptive fields,displays little supraspinal control, and is usually permanently lostfollowing severe spinal injury producing a bilateral region of areflexiabelow the level of the lesion [Borgens, R. B., et al., J. Comp. Neurol296: 634-653; Blight, A. R., et al. (1990) J. Comp. Neurol 296: 614-633;Thierault, E. and Diamond, J. (1988) J. Neurophys. 60: 446-463;Thierault, E. and Diamond, J. (1988) J. Neurophys. 60: 463-477] (FIGS.12A and 12C). In such cases, recovery of the CTM reflex in response totactile or electrical stimulation within the region of areflexia isusually not observed for the life of the animal. The anatomy,physiology, and character of the CTM behavior—both normal and inresponse to lesioning—has been reported in both rat and guinea pig[Blight, A. R., et al. (1990) J. Comp. Neurol.-296: 614-633; Thierault,E. and Diamond, J. (1988) J. Neurophys. 60: 446463; Thierault, E. andDiamond, J. (1988) J. Neurophys. 60: 463-477].

To visualize and quantify the CTM behavior, a matrix of dots was markedonto the backskin of the animal. When the shaved backskin of sedatedguinea pigs was touched with a monofilament probe, the backskin inuninjured or intact receptive fields contracted in response to thetactile stimulation (FIG. 12B). The boundary between responsive andunresponsive backskin was marked onto the backskin with a marker whilethe entire study period was videotaped from a platform-mounted cameraabove. Probing outside this area does not evoke skin contraction.

Animals were arranged on a background grid to facilitate theregistration of successive video images. Video images were acquired toan Intel® Dual Pentium® Pro computer. Superimposing of images, thecoloring of receptive field boundaries made on the backskin of theanimals during CTM testing, and the general management of video imageswas performed using Adobe® Photoshop® software. Final Plates wereconstructed with Microsoft® Powerpoint software and printed on an EpsonStylus Color 800 printer. Quantitative planimetry of the unit area ofreceptive fields—or regions of behavioral loss and recovery—was carriedout using IP Lab Spectrum™ software.

Statistics

The Mann Whitney, two-tailed test was used to compare the means of thedata derived from experimental and sham-treated groups. To compare theproportions between groups, Fishers exact test was used. All tests wereperformed using INSTAT software.

Results

The standardized injury produced a similar loss of CTM functioning inexperiments testing the response to the immediate application of PEG andexperiments testing the response to the delayed application of PEG. Thepercent loss of CTM receptive fields (FIG. 12C) was not statisticallydifferent between either of the two experiments or between sham-treatedand PEG-treated guinea pigs in either experiment (P>0.4, Student's ttest, two-tailed). Only one animal died during the course of this study.

Behavioral Loss and Recovery of the CTM Reflex

In both experiments, 19 of the 22 sham-treated animals did not recoverCTM functioning, as seen in Tables 2 and 3. TABLE 2 Percent recovery ofthe CTM¹ reflex in adult guinea pigs after immediate treatment with PEG.Day 1 Day 2 Animal Animal No. {overscore (X)} ± SEM² Range³ Stat⁴ No.{overscore (X)} ± SEM² Range³ Stat⁵ Stat⁶ Control  0/11 0 0 0.0005  2/110.18 ± 1.9 −15-11   0.015 0.006 PEG- 10/14 6.2 ± 1.4 0-15.2 10/14 13.8 ±3.8   0-42.1 Treated¹The increase in the area of backskin regaining sensitivity to tactilestimulation is given as a percent of the total region of CTM behavioralloss. All unit areas in CM2 were calculated by planimetry from capturedvideo images.²F = Mean % Recovery of the CTM Reflex and Standard Error of the Mean³The range of the control data set at 4 days includes the percentincrease in the area of CTM loss which is given as a negative number.⁴P value: proportion of recovered and unrecovered animals evaluated withFishers' exact test, two-tailed.⁵P value: means compared with Mann Whitney, two tailed test.

TABLE 3 Percent recovery of the CTM1 reflex in adult guinea pigs afterdelayed treatment with PEG. Day 1 Day 3 2 Weeks 1 Month Animal AnimalAnimal Animal Number {overscore (X)} ± SEM² Number {overscore (X)} ± SEMNumber {overscore (X)} ± SEM Number {overscore (X)} ± SEM Control 0/11 01/11 2.8 ± 2.8  1/11 2.8 ± 2.8  1/11 2.8 ± 2.8 PEG- 9/11 11.8 ± 2.9 9/1111.9 ± 2.9  10/11 15.3 ± 3.3  10/11 19.5 ± 3.02 Treated Statistic0.0002⁴ NA⁶ 0.002⁴ 0.009⁵ 0.0003⁴ 0.003⁵ 0.0003⁴ 0.0008⁵¹The increase in the area of backskin regaining sensitivity to tactilestimulation is given as a percent of the total region of CTM behavioralloss. All unit areas in CM2 were calculated by planimetry from capturedvideo images.²X = Mean % Recovery of the CTM Reflex and Standard Error of the Mean³The range of the control data set at 4 days includes the percentincrease in the area of CTM loss which is given as a negative number.⁴P value: proportion of recovered and unrecovered animals evaluated withFishers' exact test, two-tailed.⁵P value: means compared with Mann Whitney, two tailed test.⁶Statistical comparison of means not applicable to this data set.

During the first experiment, CTM functioning actually worsened by day 4in two control animals (the region of CTM loss increased by 2% and 15%respectively; Table 1). In contrast, CTM functioning recovered in 10 of14 PEG-treated animals in the first experiment (about 80%; FIG. 12D,Table 2), and in greater than about 90% of experimental animals in thesecond experiment. In all PEG-treated animals, the restored region ofCTM competent backskin was observed within the first day followingtreatment and continued to increase in size with time (Tables 2 and 3).For example, the average unit area of backskin recovering CTMsensitivity nearly doubled from about 12% (day 1) to about 20% by 1month post application in the second experiment (Table 3). Both theincreased proportion of animals recovering CTM function, and the averageincrease in the areas of recovered CTM competent backskin in response toPEG, was statistically significant (Tables 2 and 3).

EXAMPLE 6 In Vivo Effect of PEG on Conduction of Somatosensory EvokedPotentials Through Crushed Guinea Pig Spinal Cord

This example demonstrates that in vivo application of PEG to an injuredspinal cord allows for conductance of evoked CAPS, known assomatosensory evoked potentials (SSEPs), through the region that wasinjured.

Physiological Recording of SSEPs

A pair of subdermal electrodes stimulated nerve impulses from the tibialnerve of the hindleg (stimuli trains in sets of 200 at 3 Hz; stimulusamplitude less than or about equal to 3 mA square wave, 200 Psduration). Evoked volleys of CAPs were conducted into the spinal cord,projected to, and recorded from, the sensory cortex of the brain.Recording of the nerve impulses at the brain employed a pair ofsubdermal electrodes located above the level of the contralateral cortexwith reference electrodes located in the ipsilateral pinna of the ear.Stimulation, recording, signal averaging, and the computer management ofthis physiological data utilized a Nihon Kohden Neuropak 4stimulator/recorder and PowerMac G3 computer.

Measurements of SSEPs were carried out in every animal prior to spinalcord injury (FIGS. 13A-13D). In all animals (at any test period), thefailure to record an SSEP following stimulation of the tibial nerve wasfurther confirmed to be due to a lack of conduction through the spinalcord lesion by a control test carried out on the same animal. In thisprocedure, the medial nerve of the forelimb was stimulated, initiatingevoked potentials in a neural circuit unaffected by the crush injury(FIGS. 13A-13C). To perform this test, recording electrodes were left inplace while stimulating electrodes were relocated to stimulate themedian nerve using identical parameters of stimulation.

Statistics

The Mann Whitney, two-tailed test was used to compare the means of thedata derived from experimental and sham-treated groups. To compare theproportions between groups, Fishers exact test was used. All tests wereperformed using INSTAT software.

Results

Physiological Measurements of Conduction through the Spinal Cord Injury

Physiological measurements of SSEP conduction were performed in everyanimal prior to spinal cord injury and within 5-15 minutes after surgery(FIGS. 13B and 13C; FIGS. 14A-14C) to provide a basis for latercomparison. In the uninjured animal, SSEPs were typically observed tosegregate into two peaks; early arriving (latency, about 20-30 ms) and alater arriving SSEP (about 35-45 ms; FIGS. 13B and 13C; FIGS. 14A and14B). In the first experiment depicted in FIG. 13C, subsequent recordswere taken at approximately 30 minutes, 1 hour, 24 hours, and 4 daysafter PEG treatment. In the second experiment, subsequent measurementswere made approximately 6-8 hours, 18-24 hours (data not shown), 3 days,2 weeks, and 1 month following the delayed application of PEG. In allanimals, the failure to record an SSEP following stimulation of thetibial nerve was further confirmed to be due to a lack of conductionthrough the injury by a control procedure carried out on the sameanimal, where the medial nerve of the forelimb was stimulated. In allcases, this produced a characteristic SSEP for this spinal circuitunaffected by the injury (FIGS. 13A-13C;FIG. 14B). In thisinvestigation, sham-treated animals never regained the ability toconduct SSEPs through the injury site.

In the first experiment depicted in FIGS. 13B and 13C, a detectable SSEPwas recorded within a few minutes after PEG application. Quantitativeevaluation of 10 of these animal's electrical records showed that SSEPamplitudes continued to improve—averaging about 40% of their preinjurylevel, and displaying more typical latencies with time (FIGS. 13C and13D). Remarkably, within minutes of the spinal injury, the total loss ofphysiological functioning was reversed in 23 of 25 PEG-treated animals.In the two animals that did not immediately respond to PEG application,SSEP recovery was later observed at the 2 weeks time-point (FIG. 14C,Table 3). In the four animals whose recovered SSEPs were tested byreinjury, the second compression of the spinal cord at the originalinjury site completely eliminated recovered SSEPs, confirming these wereconducted through the lesion. In 9 of 11 experimental animals, thedelayed application of PEG (about 8 hours post injury) produced adetectable SSEP within 18 hours (FIG. 14C).

All 34 PEG-treated animals recovered SSEP conduction contrasted to thecomplete failure of all control guinea pigs to conduct evoked potentialsthrough the lesion. Only 3 of 22 sham-treated animals recovered CTMfunction in both experiments, while 20 of 25 PEG-treated animalsrecovered variable amounts of CTM functioning which continued to improvewith time (Table 2, 3).

SUMMARY OF THE RESULTS

This report is the first to show that an immediate and brief applicationof a hydrophilic fusogen, polyethylene glycol, to the site of a severecompression injury to the adult guinea pig spinal cord in vivo resultsin an immediate recovery of nerve impulse conduction and a progressiverecovery of behavioral functioning of the CTM reflex—a quantitativeindex of white matter integrity [Borgens, R. B., et al. (1990) J. Comp.Neurol. 296: 634-653; Blight, A. R., et al. (1990) J. Comp. Neurol. 296:614-633; Borgens, R. B., et al. (1987) Science 238: 366-369].Furthermore, an 8 hour delay in this application still resulted in asimilar recovery of these functions. In sharp contrast, sham-treatedanimals never recovered the ability to conduct nerve impulses, and theminor occurrence of spontaneous recovery of CTM function was rarecompared to the PEG-treated group.

This report provides clear evidence of a behavioral recovery dependenton an identified neural circuit within the damaged mammalian centralnervous system in response to this experimental treatment [Borgens, R.B. and Shi, R. (1999) J. Faseb (in press)]. This suggests molecularrepair and fusion of nerve membranes as a novel treatment of severetrauma to both peripheral nervous system as well as central nervoussystem tissue.

EXAMPLE 7 Polyethylene Glycol Rapidly Restores Physiological Functionsin Damaged Sciatic Nerves of the Guinea Pig

This example demonstrates the ability of a PEG application to fusesevered axons of the isolated sciatic nerve permitting an immediaterecovery of CAP conduction using recording techniques described above. Abrief application of PEG proved able to reconnect the proximal anddistal sciatic segments. In an attempt to reverse functional losssubsequent to a severe crush injury of the sciatic nerve in an in vivosciatic injury model, it was shown that PEG application can produce astatistically significant enhancement of muscle functioning subsequentto mechanical damage to its motor efferent compared to sham-treatedanimals which displayed a variable level of endogenous repair.

Materials and Methods

Removal of Sciatic Nerve for In Vitro Study

The sciatic nerves of adult female guinea pigs of 350-500 gram bodyweight were used for these experiments. The animals were deeplyanesthetized with 60-mg/kg ketamine hydrochloride and 10-mg/kg xylazinegiven intramuscularly prior to dissection of the sciatic nerve. Onceadequate anesthesia was obtained, the sciatic nerve was dissected fromits exit from the sciatic notch of the hind leg to beyond its branchinginto the tibial and peroneal nerves. Following exposure of the sciaticnerve it was gently blunt probed free from underlying fascia, and anabout 38 mm length was removed to an oxygenated vial of Krebs' solutionafter severing it at the nerve's proximal and distal extremes. Allanimal use was in compliance with State, Federal, and Universityguidelines under protocols approved by the Purdue University Animal Careand Use Committee.

In Vitro Sciatic Fusion and Electrophysiological Recordings

The isolated sciatic nerves were placed in a three compartment, doublesucrose gap recording chamber. A full description of this chamber,including diagrams and details of its construction and use, has beenpreviously reported [Shi, R., Borgens, R. B., Blight, A. R. (1999):Functional reconnection of severed mammalian spinal cord axons withpolyethylene glycol, J. Neurotrauma, 16: 727-738; Shi, R., Borgens, R.B. (1999): Acute repair of crushed guinea pig spinal cord bypolyethylene glycol, J. Neurophysiology, 81: 2406-2414]. Briefly: abouta 38 mm long segments of sciatic nerve was placed in the chambercrossing all of its three large interconnected compartments. The ends ofthe nerve were immersed in isotonic KCL (120 mM), while the centralregion was immersed in Krebs' solution (NaCl, 124 mM; KCL, 2 mM; KH₂PO₄,1.24 mM; MgSO₄, 1.3 mM; CaCl₂, 26 mM; sodium ascorbate, 10 mM; dextrose,10 mM; NaHCO₃, 26 mM; equilibrated with 95% O₂-5% CO₂). Thus the endswere maintained at approximately intracellular potential while themiddle of the sciatic nerve was maintained at approximatelyextracellular potential. Each of these three large compartments wereseparated by a small compartment of flowing sucrose (230 mM) helping tomaintain electrical isolation of the ends of the nerve and to reducemixing of the media. CAPs were evoked at one end by bipolar electrodesand recorded at the other end of the strip of spinal cord continuouslyduring each experiment. Recordings were begun after the nerve hadequilibrated within the chamber, and during and after a completetransection of the sciatic nerve within the middle compartment.

Typical physiological functioning of the nerve required about ½ to 1hour of incubation time while immersed in oxygenated Krebs' at 37° C.Once CAP propagation stablized, the sciatic nerve was completely severedwith a laboratory fabricated cutter (a shard of a razor blade attachedto an applicator stick), and the two ends of the nerve were observed tobe separated by a gap of about 1 mm with a stereomicroscope. Stimulationand recording was continued during transection which completelyeliminated the conduction of CAPs from one end to the other. Followingtransection, the two ends of the cord segments were pushed together,i.e., abutted tightly using a laboratory fabricated device that appliedgentle pressure on one segment of the sciatic nerve pressing and holdingit against the other [as in Shi, R., Borgens, R. B., Blight, A. R.(1999): Functional reconnection of severed mammalian spinal cord axonswith polyethylene glycol, J. Neurotrauma, 16: 727-738]. The device wasmounted on a micropositioner, and contacted the spinal cord parenchymawith a strip of nylon mesh stretched across two metal bands [Shi, R.,Borgens, R. B., Blight, A. R. (1999): Functional reconnection of severedmammalian spinal cord axons with polyethylene glycol, J. Neurotrauma,16: 727-738]. A solution of PEG (1800, 50% by weight in distilled water)was applied by pressure injection through a micropipette to the abuttedsegments as a continuous stream about 0.5 mm wide and continuing forabout 2 minutes. The PEG was applied to one side of the transection,washed across it, and was removed by aspiration on the other side usinga second suction pipette. During the PEG application, a continuousstream of oxygenated Krebs' solution was maintained. Theelectrophysiological properties of the fused sciatic nerve was monitoredcontinuously for approximately 1 hour.

The storage of real time digitized physiological data, management ofthis data, and the signal averaging of elicited CAP wave forms wasaccomplished using a custom designed Labview computer program on a PowerMacintosh G-3 computer.

In Situ Isolation of Sciatic Nerve

For in situ experiments, the sciatic nerve of the hind leg wassurgically exposed past its distal branches as described above. The skinwas incised and dissected away from the gastrocnemius muscle. The entiredorso-lateral aspect of the gastrocnemius muscle and its distalinsertion (Achilles' tendon) were exposed. All branches of the sciaticnerve except that to the gastrocnemius muscle were incised with irisscissors. Care was taken to irrigate the entire exposed wound frequentlyduring dissection and physiological recording with lactated Ringer'ssolution to avoid desiccation. Once the sciatic nerve and gastrocnemiusmuscle were exposed, the animal was secured to a Plexiglas platform withthe pelvis and lower limbs elevated approximately 3 cm above thestation. The elevation of the limbs enabled free passive and activeankle motion.

Electrophysiological Recording In Situ

Hook shaped AgAgCl stimulation electrodes were fabricated from 26 gaugesilver wire and with a micropositioner, gently supported the proximalsciatic nerve just as it exited the sciatic notch. Petroleum jelly wasapplied to the contact area to cover it and help insulate the point ofelectrical stimulus from the rest of the body. A paddle shapedtransducer (LAB # FT-100. CB Science Inc., Dover, N.H.) was positionedwith a micropositioner so that the paddle was firm against the distalmetacarpals of the same foot. This transducer was calibrated to measurethe force of contraction of the gasctrocnemius in dynes. One end of asliding displacement transducer (LAB #DT-475. CB Science Inc.) wasattached to the table and the slide bar sutured to the Achilles' tendonwith 3-0 silk sutures. With this arrangement, the displacement of themuscle in mms was measured during contraction. Finally, a pair of AgAgCldisc electrodes were fabricated from 20 gauge wire by heating the end ofthe bare silver wire to a molten state and pressing the tip to ice.These “disc electrodes” were then chlorodized by conventionaltechniques. The pair with electrode spacing of about 2-3 mm was placedon the belly of the gastrocnemius muscle to measure compound muscleaction potentials (APs in mVs) subsequent to electrical stimulation ofthe sciatic nerve (FIG. 1).

Following the application of all the electrodes and transducers, thesciatic nerve was stimulated with square wave pulses (≦6 Hz, 1 msduration) from the integral stimulator of the LAB™ computer managedintegrated system for physiological measurement and recording(PowerLab/4S; ETH-400 bridge amplifier, CB Science Inc. Dell optiplexGX1p computer and CHART™ software, AD Instruments). The leastsuprathreshold voltage required producing maximal force and displacementresponse of the muscle was determined, and 1.25 times that stimulusvoltage was used for the remainder of each individual experiment

Nerve Injury and PEG Application In Situ

Following the exposure of the nerve and muscle and the arrangement ofthe stimulation and recording electrodes, baseline values of the force,displacement, and muscle action potentials were established. Then, thesciatic nerve was crushed for 90 seconds with modified Dumont 5 forceps.The forceps had been filed so that the tip width was 1.5 mm, and theywere bent so that the tips were parallel to each other. Preliminaryexperiments were used to determine the duration of a standarddisplacement crush to completely eliminate all three functional measuresfor a minimum of ½ hour, allowing only minimal recovery of any onefunctional test within 1-1{fraction (1/2)} hour post injury (data notshown). Prior to, and immediately following the crush injury, a baselinerecord of all functional responses to sciatic stimulation was obtainedfor all animals. Subsequently, 0.05-0.1 cc of a PEG or control solutionwas injected beneath the epineurium at the region of crush injury with a29 gauge needle on an insulin syringe. A vital dye was included in eachsolution to enable direct observation of its removal. Each solution wasleft in place for 2 minutes. The epineurium was then openedlongitudinally with a razor, and the solution was irrigated away withlactated Ringer's. Electrophysiological recording was performed at5-minute intervals for 90 minutes after administration of each solution.The animals were then euthanized by intracardiac injection of 50 mgpentobarbital.

Those animals treated within 10 minutes of injury were divided into thefollowing groups: (1) the PEG treated group, N=8; (2) a Krebs' solutioncontrol group, N=6; and (3) a distilled water control group, N=6. A 50%by weight solution of 1800 MW PEG in distilled water was used in thetreated group. Krebs solution consisted of NaCl 124 mM, KCl₂ mM, KH₂PO41.2 mM, MgSO₄ 1.3 mM, CaCl₂ 1.2 mM, dextrose 10 mM, NaHCO₃ 26 mM, andsodium ascorbate 10 mM. The data obtained from both control groups(described below) were pooled since there was no statistical (orbehavioral) difference between them.

Nerve Injury and Delayed PEG Application

Delayed application of PEG or a Krebs' control solution was performed 4hours after crush injury. Six guinea pigs were treated with PEG (50%solution of 1800 MW PEG), and six animals were treated with the Krebs'control solution. The animals were anesthetized as described previously.Then, a 1 cm segment of the sciatic nerve was exposed at themid-hamstrings level, and a 90 second crush was performed with themodified Dumont 5 forceps. The wound was irrigated with lactatedRinger's and closed with 3-0 silk sutures. The animals were kept under awarming lamp. Three and a half hours later, the animal wasre-anesthetized. The entire sciatic nerve and gastrocnemius muscle wereexposed, and the stimulating and recording electrodes and transducerswere arranged as described above. Four hours after the crush injury, thesolution of either PEG or Krebs' was administered beneath the epineuriumat the injury site and was removed after 2 minutes by the techniquesdescribed above. Electrophysiological recording was performed at5-minute intervals for 60 minutes following administration of eachsolution. The animals were then euthanized by intracardiac injection of50 mg pentobarbital.

Statistics

Population means were compared with Mann—Whitney two tailed test, whileproportions were compared with Fishers exact test, two tailed.

Results

Fusion of Severed Sciatic Nerve In Vitro

Our investigation began with determining if any axons within the twosegments of a severed sciatic nerve were able to be immediately reunitedby PEG application. Four sciatic nerve (lengths about 38 mm) were testedin this way in the double sucrose gap chamber. Each was allowed to“recover” from the isolation procedure for varying times up to one houruntil the normal capacity to conduct compound action potentials from thepoint of stimulation to the recording site on the other end of thechambers had stabilized. The average CAP magnitudes prior to the injurywere 5, 3.5, 1.5, and 4.8 mV respectively. Stimulation and recording ofCAPs was continued during the process of transection, simultaneous withthe total elimination of CAP conduction. Within 15 minutes of themechanical abutting of the proximal and distal segments and 2-minuteapplication of PEG, CAP conduction was variably restored in all foursciatic nerves tested, with an average recovery of 3.45% of thepretransection amplitude. FIG. 16 shows electrical records one suchsciatic nerve tested. In summary, all four attempts to fuse completelysevered sciatic nerves were successful, restoring a variable level ofnerve impulse conduction through the lesion.

Spontaneous and PEG Mediated Recovery of Crushed Sciatic Nerve In Situ

Immediately after the crush procedure, the gastrocnemius muscle did notshow any response to stimulation of the sciatic nerve in all animals.FIGS. 17A-17C, 18A, 18B, and 19 show recordings of all three functionaltests prior to injury to the nerve, and the complete loss of these asrevealed by physiological recordings begun immediately after the injury.FIGS. 17A-17C also shows the lack of response to stimulation 1 hourlater in a control animal. This was typical of sham-treated animals.Only three of the 12 animals in this group recovered any one of thefunctional tests by 1 hour post-treatment.

In contrast, 7 of 8 PEG-treated animals recovered at least one of thesefunctions within the first 35 minutes post injury. Four of these animalsrecovered at least one measure by 5 minutes post treatment, another at10 minutes, and two more by 30 minutes. This difference in proportionbetween controls and PEG-treated animals was statistically significant(P=0.019, Fishers exact test). In the PEG-treated group, 1 of the 8recovered all three functional measures by 1 hour post treatment, 3 ofthe 8 recovered two of them, and the balance (4 of 8) recovered onemeasure of functional recovery as mentioned above. FIGS. 18A and 18Bshow recordings of the three tests prior to injury and their eliminationby the crush injury to the sciatic similar to FIGS. 17A-17C but in ananimal prior to treatment with PEG. FIG. 19 documents the recovery oftwo of the three functional measures (muscle action potential and forceof muscle contraction) within the first 5 minutes after PEG applicationin this animal. The response was robust necessitating a reduction in thesensitivity of recording. FIGS. 20A-20C show the only animal (which wasPEG-treated) to recover all three indices of functional recovery, thoughthere was some indication that the muscle AP was not completelyeliminated following injury when the sensitivity of the recording wasincreased 10 fold in this one case. Overall the most sensitive andconsistent indicator of recovery in all animals was the measurement ofmuscle contraction force—while the least sensitive was the recording ofa measurable displacement of the hind foot. The latter test was droppedfrom the regimen when delayed PEG applications were evaluated (describedbelow). Table 4 provides a summary of these data. TABLE 4 Responses toImmediate Application of PEG Post Animal#¹ Pre injury² injury³ 5 min⁴ 15min⁴ 30 min⁴ 60 min⁴ Statistic⁵ Control 12 12/12  0/12  1/12 — 1/12 1/12P = 0.019 PEG- 8 8/8 0/8 4/8 1/8  2/12⁶ — treated¹Animal Number²Number possessing measurable functional responses to sciaticstimulation over total tested (see methods)³Number possessing measurable functional responses to sciaticstimulation subsequent to sciatic injury over total tested⁴Elapsed time to recovery after application; number over total tested⁵Statistical comparison of totals; Fisher Exact test, two tailed⁶One of these preparations recovered at about 35 minutes.

Due to the all or none character of muscle excitability, a comparison ofthe mean peak AP amplitudes would not have been as informative as wouldbe the proportion of animals recovering excitability following nerveinjury and treatment. Comparison of the mean force of muscle contractionhowever provided a way to compare the relative degree of muscle“recovery” between the groups. The mean contraction force in dynes wassignificantly improved by the PEG treatment (X=23835±19991; range0-163000) compared to Controls (X=433+276.3; range=0-2880. P=0.008,Mann-Whitney two tailed test).

No difference (behavioral or statistical) was detected between thegroups when a 1 hour delayed application of PEG was tested (data notshown). Comparison between sham-treated and PEG-treated animalsfollowing a 4 hour delayed application was informative however—revealinga capability of PEG to still be able to improve functional outcomes. Theproportion of Control animals (2 of 6) showing a recovery of at leastone functional measure four hours post injury did not change subsequentto the sham application. Following PEG application however, this numberquadrupled (1 of 6 animals showed spontaneous recovery by the time ofthe 4 hour application, which improved to 4 of 6 within 5 to 45 minutesof the treatment). Due to the reduced number of animals this increase inproportion relative to controls did not reach significance, but showed astrong trend in this direction (P=0.06, Fishers' exact test).

Discussion

Using both an in vitro and in vivo assessment of physiologicalfunctioning in cut and crushed sciatic nerve, it was discovered that abrief (about 2 minute) topical application of PEG is sufficient torestore variable levels of electrophysiological conduction through thelesion. This was demonstrated by PEG mediated reconnection of completelysevered sciatic nerves maintained within a double sucrose gap recordingchamber, and an in vivo evaluation of muscle excitability following asevere proximal crush injury to its motor efferent. In the latter, twoother indices of muscle responsiveness were simultaneously monitored,the force of gastrocnemius contraction and the displacement of themuscle following electrical stimulation of the sciatic nerve. Asdescribed, muscle displacement was the most crude, and least sensitivemeasure of functional recovery. The measure of recovering muscle APs wasan intermediate success. Sometimes APs could not be measured traversingthe muscle preparation even though simultaneous gastrocnemiuscontraction was visible and measurable with the force transducer. Thiswas not completely unexpected given the difficulty of measuringextracellular APs in a blood field.

In both testing procedures, PEG application resulted in a rapid recoveryof functioning at a time when control preparations had not spontaneouslyrecovered. This was easiest to detect when PEG was applied withinminutes of the injury in situ, though a 4-hour delayed application ofPEG greatly improved the percentage of cases recovering at least onefunctional measure. The reduced number of animals studied at these latertimes did not allow significance to be reached though a strong trendtowards significance was noted.

Mechanical Damage to the Axon and its Reversal by PEG

It is now clear that in response to mechanical damage to nervemembranes, the instantaneous and primary insult is a local breakdown ofthe ability of the membrane to act as an ionic fence.Electrophysiological conduction is impaired or eliminated by thesustained collapse of the membrane potential at the site of damagecaused by the unregulated exchange of ions—principally Potassium andSodium. Anatomical integrity of the axon is compromised by the localincrease in Calcium entering cytosol at the foci of damage. Thisincrease in intracellular Ca⁺⁺ from unregulated entry into the cell isalso exacerbated by rising levels of intracellular Na⁺, which initiatesrelease of Ca⁺⁺ from intracellular stores [Carafoli, E., Penniston, J.(1985): The Calcium Signal, Sci. Am., 253: 70-78; Rosenberg, L., Lucas,J. (1996): Reduction of NaCl increases survival of mammalian spinalneurons subjected to dendrite transaction injury, Brain Research, 734:349-353; Lucas, J., Emery, D., Rosenberg, L. (1997): Physical injury ofneurons: Important roles for sodium and Chloride ions, TheNeuroscientist, 3: 89-111]. The net result is the depolymerization ofthe cytoskeleton, the activation of various Ca⁺⁺ dependent intracellularcatabolic enzymes, and other biochemistries leading to local progressivecellular dissolution, axotomy in the most extreme cases, anddegeneration of the distal axonal segment in mammals. The duration ofthis process is of course variable, depending on many characteristics ofthe nervous tissue damaged and the severity of the impact, compression,or stretch—however it may require many hours to days to run it's course.It is a working hypothesis that this process can be greatly reduced inscope, through the acute use of hydrophilic polymers that can seal eventhe worst breaches to axonal membrane. This can be demonstrated by theability of PEG to actually fuse proximal and distal segments of axonsrestoring anatomical and physiological functioning [these data and Shi,R., Borgens, R. B., Blight, A. R. (1999): Functional reconnection ofsevered mammalian spinal cord axons with polyethylene glycol, J.Neurotrauma, 16: 727-738]. In the less severe injuries to the axolemma,the action of the polymer likely enhances the natural reparativemechanism of sealing by axons. Natural or spontaneously sealing of crushinjury to the sciatic nerve, and resultant functional recovery, wasdemonstrated in this study. This recovery was enhanced by PEGapplication. In the most severe cases of mechanical damage to axons,PEG-mediated sealing likely prevents axotomy, immediately permitting APconduction through the injury site, and variable levels of immediatefunctional recovery dependent on it. Below the experimental evidencesupporting these notions is set forth in detail.

The Action of PEG on Damaged Membranes

There are several hypotheses concerning the ability of large hydrophilicpolymers like PEG and its cousins, the triblock polymers or non-ionicdetergents to reverse cell permeabilization. In certain cases, and atlower molecular weights, PEG may have detergent like properties similarto amphipathic polymers (poloxamers and polaxamines). These may formthin micelle films covering the breach in the membrane. In the triblockpolymers, the hydrophobic “head” of the molecules may actually insertitself into the breach in the membrane, since the hydrophobic core ofthe plasmalemma is exposed by the injury. The hydrophilic PEG “tails”integrate with the outer leaflet. It is also possible that PEG may sealporated membranes through acute dehydration of the local region where itis applied. This is envisioned to enable the structural elements of themembrane (proteins, glycolipids, etc) to resolve into each other as thepolar forces arising from the aqueous phase (helping to maintain theirorganization within the membrane) is now absent or reduced. When PEG isremoved and the local membrane(s) are rehydrated, spontaneous reassemblyof these structural elements leads to a restoration of the membrane. Thelatter scenario helps explain the immediate recovery of excitabilityfollowing topical PEG treatment to nerve membranes, and the success ofonly brief applications of the polymer to injured tissues. Theseputative mechanisms of action underlying the polymer mediated fusion andrepair of traumatized cell membranes have been previously discussed[Shi, R., Borgens, R. B. (1999): Acute repair of crushed guinea pigspinal cord by polyethylene glycol, J. Neurophysiology, 81: 2406-2414;Borgens, R. B., Shi, R. (2000): Immediate recovery from spinal cordinjury through molecular repair of nerve membranes with polyethyleneglycol, FASEB, 14: 27-35; Lentz, B. R. (1994): Induced membrane fusion;potential mechanism and relation to cell fusion events, Chem. and Phys.of Lipids, 73: 91-106; Lee, J., Lentz, B. R. (1997): Evolution of lipidstructures during model membrane fusion and the relation of this processto cell membrane fusion, Biochemistry, 36: 6251-6259; Merchant, F. A.,Holmes, W. H., Capelli-Schellpfeffer, M., Lee, R. C., Toner, M. (1998):Poloxamer 188 enhances functional recovery of lethally heat-shockedfibroblasts, J. Surgical Research, 74: 131-140].

In general, use of these families of high molecular weight polymersrepresent a new concept, as well as a practical means, to deal withacute trauma to tissues caused by the primary breakdown of cellmembranes. This includes a reversal of cell permeabilization in variousand different injury models including electric shock myonecrosis [Lee,R. C., River, L. P., Pan, F. S., Ji, L., Wollman, R. S. (1992):Surfactant-induced sealing of electropermeabilized skeletal musclemembranes in vitro. P.N.A.S. 89: 4524-4528], testicular reperfusioninjury [Palmer, J. S., Cromie, W. J., Lee, R. C. (1998): Surfactantadministration reduces testicular ischemia-reprefusion injury, JUrology, 159: 2136-2139], heat shock mediated cell death [Padanlam, J.T., Bischof, J. C., Cravalho, E. G., Tompkins, R. G., Yarmush, M. L.,Toner, M. (1994): Effectiveness of Poloxamer 188 in arresting calceinleakage from thermally damaged isolated skeletal muscle cells, Ann N.Y.Acad. Sci,. 92: 111-123], and radiological damage to cells [Hannig, J.,Yu, J., Beckett, M., Weichselbaum, R., Lee, R. C. (1999): Poloxamine1107 sealing of radiopermeabilized erythrocyte membranes, Int. J.Radiat. Biol., 75: 379-385].

Repair of Nervous System Damage with PEG

The ability of a hydrophilic polymer, PEG, to fuse and seal the axolemmasubsequent to mechanical damage was tested. Studies first used ventralspinal cord white matter isolated from adult guinea pigs. Strips ofspinal cord were maintained in the double sucrose gap recording chamberand completely transected—then fused with PEG [Shi, R., Borgens, R. B.,Blight, A. R. (1999): Functional reconnection of severed mammalianspinal cord axons with polyethylene glycol, J. Neurotrauma, 16:727-738], or crushed within the chamber and repaired with PEG [Shi, R.,Borgens, R. B. (1999): Acute repair of crushed guinea pig spinal cord bypolyethylene glycol, J. Neurophysiology, 81: 2406-2414]. In both ofthese cases, a topical, 2 minute application of the polymer (about1400-1800 Daltons) rapidly restored physiological conduction of compoundaction potentials through the lesion within minutes following injury.Moreover, intracellular labeling with two different fluorescent markers(rhodamine and flourescein labeled dextrans) demonstrated thatphysiological functioning following transaction of spinal cord and PEGtreatment was accompanied by restored anatomical integrity of axonsacross the transaction plane by the physical reattachment of theirproximal and distal segments [Shi, R., Borgens, R. B., Blight, A. R.(1999): Functional reconnection of severed mammalian spinal cord axonswith polyethylene glycol, J. Neurotrauma, 16: 727-738].

The ability of PEG to repair a severe standardized compression of thespinal cord in vivo has also been tested. The research usedextracellular stimulation of the tibial nerve of the hind leg and therecording of volleys of CAPs arriving at the contralateral sensorimotorcortex (so called somatosensory evoked potentials or SSEPs) as an indexof electrophysiological recovery, and the recovery of the CutaneusTrunchi Muscle Reflex (CTM) as a index of behavioral recovery subsequentto severe spinal cord injury. Spontaneous recovery from spinal cordinjury through natural mechanisms of repair occurs in less than 20% ofthe animals followed for a minimum of 1-month post injury. A 2 minutePEG application to the exposed spinal cord injury immediately after thecrush (or delayed for 7-8 hours) results in a recovery of the CTM reflexin over 90% of the treated population. A standardized lesioningtechnique [Moriarty, L. J., Duerstock, B. S., Bajaj, C. L., Lin, K.,Borgens, R. B. (1998): Two and three dimensional computer graphicevaluation of the subacute spinal cord injury, J. Neurologic. Sci., 155:121-137] also results in a complete loss in SSEP conduction in thespinal cord in 100% of the injured animals, and there was no recovery ofconduction through the lesion observed in any of these Controltreatments. A striking and unexpected result was the recovery of SSEPconduction in 100% of PEG treated animals, usually between a few hoursto one-day post treatment [Borgens, R. B., Shi, R. (2000): Immediaterecovery from spinal cord injury through molecular repair of nervemembranes with polyethylene glycol, FASEB, 14: 27-35]. By using a dyeexclusion test, it was also learned that this brief PEG applicationindeed seals spinal cord axons [Shi, R., Borgens, R. (2000): Molecularrepair of Nerve membranes in crushed mammalian spinal cord withpolyethylene glycol, J. Neurocytology (in press)]. Brief exposure ofacutely injured isolated guinea pig white matter to a solution ofhorseradish peroxidase lead to endocytosis of the label marking onlydamaged axons. Most axons in the crushed spinal cord took up the label.A striking and statistically significant reduction in HRP uptake wasassociated with a 2 minute PEG treatment prior to HRPexposure—demonstrating the polymer sealed these breaches [Shi, R.,Borgens, R. (2000): Molecular repair of Nerve membranes in crushedmammalian spinal cord with polyethylene glycol, J. Neurocytology (inpress)].

Clinical Relevance

This ability of PEG to functionally reconnect severed axons is the mostchallenging test of its reparative capability. In the sciatic nerve, theorganization of the nerve trunk into fascicles and the tough surroundingepineureum raised the possibility that fusion of even some of the axonsinside may not be possible—particularly given the possibility of theirretrograde degeneration away from the plane of the transection with thefacicles. It was discovered that sciatic nerve was similar to spinalcord white matter in that each attempt to fuse some axons within the cutnerve was successful. In spinal cord this has only limited importance toclinical injuries because spinal transection is rare. However this is ameaningful result in the context of neurosurgical reattachment ofseverely damaged peripheral nerves where the ends of damaged nervetrunks may be resected prior to fascicular alignment and suturing.Additional treatment with PEG requires that procedures to stabilize theperhaps delicate fused regions for an undetermined period of time willbe necessary. This additional therapy may pay dividends in permitting alarger level of immediate functional return while eliminating variabledegrees of Wallerian degeneration and the atrophy of muscle. A decisionwas made to begin such exploration with the non-survival proceduresreported here—as long term (days to weeks) monitoring of animals withperipheral nerve injuries is complicated. Comparison of any functionaltesting is made problematic by the robust spontaneous repair andregeneration of rodent peripheral nerves. These data are indicative thata careful evaluation of the long term functional repair of peripheralnerves by PEG, and to define the critical window in time when theapplication is required, is indeed the next step in theseinvestigations. With respect to spinal cord repair using PEG, thesetechniques have now been moved into clinical testing using naturallyoccurring cases of neurologically complete paraplegia in dogs [seeBorgens, R. B., Toombs, J. P., Blight, A. R., McGinnis, M. E., Bauer, M.S., Widmer, W. R., Cook, Jr., J. R. (1993): Effects of applied electricfields on clinical cases of complete paraplegia in dogs, J. RestorativeNeurology and Neurosci., 5: 305-322; Borgens, R. B., Toombs, J. P.,Breur, G., Widmer, W. R., Water, D., Harbath, A. M., March, P., Adams,L. G. (1999): An imposed oscillating electrical field improves therecovery of function in neurologically complete paraplegic dogs, J.Neurotrauma, 16: 639-657; Blight, A. R., Toombs, J. P., Bauer, M. S. andWidmer, W. R. (1991): The effects of 4-aminopyridine on neurologicaldeficits in chronic cases of traumatic spinal cord injury in dogs: aphase I clinical trial. J. Neurotrauma. 8: 103-119].

EXAMPLE 8 Rapid Recovery from Spinal Cord Injury FollowingSubcutaneously Administered Polyethylene Glycol

This example demonstrates that a biomembrane fusion agent, specificallythe hydrophilic polymer PEG, can be safely introduced into thebloodstream by several routes of administration, and that theadministered PEG specifically targets a hemorrhagic contusion of anadult guinea pig spinal cord. A single subcutaneous injection (30%weight by weight in sterile saline) made 6 hours after spinal injury wassufficient to produce a rapid recovery of CAP propagation through thelesion, accompanied by a significant level of behavioral recovery inonly PEG-treated animals.

The results of these tests demonstrate (1) that PEG specifically targetsthe spinal cord contusion independent of whether it is applied directlyto the exposed spinal injury, or by intravenous or subcutaneousinjection, and (2) that a single subcutaneous injection of PEGapproximately 6 hours after severe SCI is sufficient to induce a rapidreversal of functional losses in nearly all PEG-treated adult guineapigs compared to the persistence of these deficits in nearly allsham-treated animals. The intravascular delivery of PEG for purposes oftreating and repairing injured nerve tissue has also been investigatedin a clinical setting using naturally produced cases of paraplegia indogs, as discussed hereinafter.

Drawing FIG. 13A: Behavioral Model and Physiological Evaluation

This drawing shows the neural circuit of the Cutaneus Trunchi Muscle(CTM) reflex, and its interruption by spinal injury. Nociceptive sensoryreceptors in the skin project their axons into the spinal cord at eachvertebral segment bilaterally via the Dorsal Cutaneus Nerves. Thesesynapse within the spinal cord and project 2nd order ascending sensorynerves in the ventral funiculus of the white matter to the cervicalregion where these synapse on bilaterally organized constellations ofCTM motor neurons. CTM motor neurons project their axons out of the cordon right and left sides via the brachial plexus, where these innervatethe cutaneous muscle of the skin via the lateral thoracic branch of theplexus. When the spinal cord is intact, tactile stimulation of the backskin within the CTM receptive field causes a rippling contraction of theskin. Stimulation outside the receptive fields of back skin does notresult in skin contractions. A spinal cord injury (drawn on only theleft side of the cord for descriptive purposes) interrupts the ascendingleg of this circuit producing a region of skin areflexia ipsilateral tothe injury and on the same side. Tactile probing within this region doesnot produce CTM contractions, usually for the life of the animal.Stimulation of back skin above the level of this unilateral lesion, oron the right side produces CTM contractions, as these receptive fieldsremain unaffected by the unilateral injury to the left side of thespinal cord.

Methods

Animal Surgery and Spinal Cord Injury

Adult Guinea Pigs (<300 gm) were anesthetized with an intramuscularinjection of 100 mg/kg ketamine HCL and 20 mg/kg xylazine and the spinalcord exposed by dorsal laminectomy. The midthoracic cord was crushedwith special blunted forceps possessing a détente. This standardized,constant displacement injury [Moriarty, L. J., Duerstock, B. S., Bajaj,C. L., Lin, K., and Borgens, R. B. (1998) Two and three dimensionalcomputer graphic evaluation of the subacute spinal cord injury, J.Neurologic. Sci., 155, 121-137] has produced more consistent anatomicalinjury to the cord and more consistent behavioral loss between animalsthan constant impact injuries (such as those produced by the variousweight drop techniques). Animals were euthanized by deep anesthesiafollowed by perfusion/fixation. The localization of an FITC decoratedPEG (Fl-PEG) in spinal cord was determined by killing the animals forhistological processing approximately 24 hours after the application orinjection of Fl-PEG. The spinal cords were dissected from the animals,and the segments of spinal cord containing the sites of injury and anintact, more rostral, segment were sectioned with a freezing microtomeand evaluated with a fluorescent microscope. Histological cross sectionswere 5 μm thick, and observed on an Olympus Van Ox Fluorescentmicroscope using excitation wavelengths of 495 and 545 nm and barrierfilters of 475 and 590 nm, respectively. Digital images were captured tothe computer with an Optronics DEI 750 camera.

To test the effects of subcutaneous injections of PEG, adult guinea pigswere anesthetized and their mid-thoracic spinal cords were surgicallyexposed and then crushed by a standardized technique. [Blight, A. R.(1991): Morphometric analysis of a model of spinal cord injury in guineapigs, with behavioral evidence of delayed secondary pathology, J.Neurolog. Sci., 103: 156-171.] Twenty animals were divided into equalgroups of 10. One group received a single subcutaneous injection of PEG(1400 MW) beneath the skin of the neck (0.5 cc; 30% in sterile lactatedRinger's solution; SLR). The sham-treated control group received asingle injection of the carrier, lactated Ringer's. Only this onesubcutaneous injection per animal was made approximately 6 hours afterthe spinal cord injury. CTM testing and SSEP recordings were carried outon all 20 animals prior to spinal cord injury, 1 day, 1 week, 2 weeks,and 4 weeks post injury.

Tracing the Distribution of PEG in Injured Spinal Cord

The FITC decorated PEG (about 1400 Daltons; prepared by MolecularProbes, Chatsworth, Calif.) was used to trace the distribution of PEGfollowing different routes of administration. Fl-PEG, 50% weight byweight in SLR was applied directly to exposed spinal cord injury site(with the dura removed) using a Pasteur pipette in two animals. As inprior experiments [Borgens, R. and Shi, R. (2000) Immediate recoveryfrom spinal cord injury through molecular repair of nerve membranes withpolyethylene glycol, FASEB 14, 27-35], PEG was removed by aspiration andthe region irrigated with SLR two minutes later. Subcutaneous injectionof 1 cc Fl-PEG (30% w/w in SLR) was made beneath the skin of the neck intwo spinal injured guinea pigs using a 22-gauge needle. For IVinjection, the jugular vein was surgically exposed, and 1 cc of FL-PEGwas 7injected using a 26-gauge needle. PEG, 30% in lactated Ringer's wasalso administered by intraperitoneal injection in one case.

In Vivo Conduction Studies

Functional deficits produced by SCI are largely caused by the loss ofnerve impulse conduction through mechanically damaged tracts of nervefibers in spinal cord white matter [Blight, A. R. (1993) Remyelination,Revascularization, and Recovery of Function in Experimental Spinal CordInjury, Advances in Neurobiology: Neural Injury and Regeneration (Seil,F. J. Ed.), Vol. 59, pp. 91-103, Raven Press, New York]. Accordingly,the loss and recovery of compound action potential (CAP) conductionthrough the spinal cord injury was evaluated by evoked potentialtechniques (somatosensory evoked potential testing or SSEP). Stimulationof the Tibial nerve of the hind limb produced ascending volleys of nerveimpulses recorded at the contralateral sensory cortex of the brain.These were eliminated between the site of stimulation and recording bythe spinal lesion—immediately abolishing the recording of these peaks(postcrush records). Each electrical record was comprised of a stimulustrain of 200 stimulations (<2 mA square wave, 200 μs duration at 3 HZ).Three sets of these recordings were made at each measurement period andaveraged to produce the single waveform presented in the following data.The appearance of original records prior to computer averaging can befound in prior reports [Borgens, R. and Shi, R. (2000) Immediaterecovery from spinal cord injury through molecular repair of nervemembranes with polyethylene glycol, FASEB 14, 27-35]. Conduction ofnerve impulses through a median nerve circuit following stimulation ofthe median nerve of the forelimb (unaffected by the spinal cord injuryat the midthoracic level) was a control procedure during SSEP recording.This control stimulation regimen was carried out in every circumstancewhere a failure to record evoked potentials at the cortex occurred inresponse to hind limb tibial nerve stimulation—to eliminate thepossibility these failures were “false negatives”. SSEP recording andaveraging was performed with a Nihon Kohden Neuropak 4stimulator/recorder and a PowerMac G3 computer. Computation of the areabeneath the early arriving SSEP peak (P1) was accomplished by scribing areference line beneath the base of the peak, and determining the unitarea contained within it as pixels using IP Lab Spectrum software.

Behavioral Studies

As an index of behavioral recovery, evaluations are made of a spinalcord dependent contraction of back skin in animals—the Cutaneus TrunchiMuscle reflex (CTM)[Blight, A. R., McGinnis, M. E., and Borgens, R. B.(1990): Cutaneus trunci muscle reflex of the guinea pig, J. Comp.Neurol., 296, 614-633; Borgens, R. B. (1992): Applied Voltages in SpinalCord Reconstruction: History, Strategies, and Behavioral Models, inSpinal Cord Dysfunction, Volume III: Functional Stimulation, (Illis, L.S. ed.), Chapter 5, pp. 110-145, Oxford Medical Publications, Oxford].The loss of CTM behavior following injury to the spinal cord is observedas a region of back skin, which no longer responds, by muscularcontraction to local tactile stimulation [Blight, A. R., McGinnis, M.E., and Borgens, R. B. (1990): Cutaneus trunci muscle reflex of theguinea pig, J. Comp. Neurol., 296, 614-633; Borgens, R. B. (1992):Applied Voltages in Spinal Cord Reconstruction: History, Strategies, andBehavioral Models, in Spinal Cord Dysfunction, Volume III: FunctionalStimulation, (Illis, L. S. ed.), Chapter 5, pp. 110-145, Oxford MedicalPublications, Oxford; Borgens, R. B., Blight, A. R., and McGinnis, M. E.(1990): Functional recovery after spinal cord hemisection in guineapigs: The effects of applied electric fields, J. Comp. Neurol., 296,634-653; Borgens, R. B., Blight A. R., and McGinnis M. E. (1987):Behavioral recovery induced by applied electric fields after spinal cordhemisection in guinea pig, Science, 238, 366-369]. This areflexia doesnot recover for the life of the animal if the relevant (and identified)ascending CTM tract is severed within the ventral funiculus as thecomplete neural circuit underlying this behavior has been identified[Blight, A. R., McGinnis, M. E., and Borgens, R. B. (1990): Cutaneustrunci muscle reflex of the guinea pig, J. Comp. Neurol., 296, 614-633].Following a severe bilateral crush injury of the mid-thoracic spinalcord (such as used here), a bilateral region of areflexia of back skinis produced that still shows very limited ability to spontaneouslyrecover [Borgens, R. and Shi, R. (2000): Immediate recovery from spinalcord injury through molecular repair of nerve membranes withpolyethylene glycol, FASEB, 14, 27-35; Borgens, R. B. (1992): AppliedVoltages in Spinal Cord Reconstruction: History, Strategies, andBehavioral Models, in Spinal Cord Dysfunction, Volume III: FunctionalStimulation. (Ilis, L. S. ed.), Chapter 5, pp. 110-145, Oxford MedicalPublications, Oxford]. A variable region of back skin recovery occurs inresponse to crush injury in a relatively small proportion of spinalinjured animals (we estimate <15% rate of overall recovery in untreatedanimals based on over a decade of experience using this reflex as anindex of white matter integrity). Furthermore, there is no compensatorysprouting of cutaneous innervation into non-functioning receptive fieldswhich might mimic a centrally mediated recovery of CTM function as theseregions of skin are not denervated [Blight, A. R., McGinnis, M. E., andBorgens, R. B. (1990): Cutaneus trunci muscle reflex of the guinea pig,J. Comp. Neurol., 296, 614-633; Borgens, R. B., Blight, A. R., andMcGinnis, M. E. (1990): Functional recovery after spinal cordhemisection in guinea pigs: The effects of applied electric fields, J.Comp. Neurol., 296, 634-653]. Complete details of the anatomicallyidentified circuit, its physiology, behavioral loss and monitoring, andother testing of the CTM as a spinal cord injury model can be found inprevious reports [Blight, A. R., McGinnis, M. E., and Borgens, R. B.(1990): Cutaneus trunci muscle reflex of the guinea pig, J. Comp.Neurol., 296, 614-633; Borgens, R. B. (1992): Applied Voltages in SpinalCord Reconstruction: History, Strategies, and Behavioral Models, inSpinal Cord Dysfunction, Volume III: Functional Stimulation, (Illis, L.S. ed.), Chapter 5, pp. 110-145, Oxford Medical Publications, Oxford;Borgens, R. B., Blight, A. R., and McGinnis, M. E. (1990): Functionalrecovery after spinal cord hemisection in guinea pigs: The effects ofapplied electric fields, J. Comp. Neurol., 296, 634-653; Borgens, R. B.,Blight A. R., and McGinnis M. E. (1987): Behavioral recovery induced byapplied electric fields after spinal cord hemisection in guinea pig,Science, 238, 366-369].

Evaluations were not made of walking, inclined plane performance, ropeclimbing, or other direct or indirect measures dependent on thefunctioning of hind limbs in spinal injured rodents. These tests aremore subjective in interpretation, are not based on identified neuralcircuits, and cannot sufficiently discriminate movements dependent onintact bilateral hind limb reflexes from those based on restoredfunctioning of damaged white matter tracts.

Statistics

Comparison of the proportions of animals in each group was carried outusing Fisher's exact test, two tailed; and a comparison of means withMann Whitney non parametric two tailed test on Instat software.

Results

FITC-Labeled PEG in Spinal Cord

Very localized regions of spinal cord tissue surrounding blood vesselsand capillaries were faintly marked in uninjured spinal cord rostral orcaudal of the injury—nearly at the level of detection (FIG. 21A). Thisfaint labeling was evident around larger vessels of the gray matter andthose associated with the pial surface. Crushed regions of spinal cordwere heavily labeled in all animals independent of the means of Fl-PEGadministration. Furthermore, this intense labeling of spinal cordparenchyma was confined to the region of contused gray and white matterbut did not extend into adjacent, intact, spinal cord parenchyma (FIG.21, B-D). In summary, PEG specifically labeled the spinal cord lesionbut not undamaged tissues of adjacent regions.

PEG Mediated Recovery of Conduction

Prior to the crush injury of the spinal cord, tibial nerve evoked SSEPsusually segregate into an early and late arriving peaks of CAPs recordedfrom the sensory cortex (P1 and P2) [Borgens, R. and Shi, R. (2000):Immediate recovery from spinal cord injury through molecular repair ofnerve membranes with polyethylene glycol, FASEB, 14, 27-35]. As in priorexperiments these peaks are completely eliminated following a severeconstant displacement crush to the midthoracic spinal cord (FIG. 4).

During the 1 month of observation following a single injection of PEG oran injection of carrier in Control animals, not one control animalrecovered the ability to conduct CAPs through the lesion as measured bySSEP recording compared to a variable recovery of CAP magnitudesrecorded to arrive at the sensorimotor cortex in 100% of the PEG-treatedanimals (P=0.0001; Fishers Exact two-tailed test; FIG. 22B; Table 5).TABLE 5 Area: CAP(P1) Area: CAP(P1), Treat- # of % loss, area % CTM #CTM # SSEP pre-injury in Post-injury Ment Animals of Areflexia¹Recovered² Recovered² Recovered³ pixels⁴ in pixels⁴ Stat⁵ PEG 10 43.6 ±0.03 32.7 ± 7.5 7/10 10/10 17026 ± 258 11482 ± 144 P = 0.14 Cont 10 42.5± 0.02 0/10  0/10 P = 0.8⁶ P = 0.003⁷ P = 0.001⁶¹The % loss of the CTM receptive field = unit area of areflexia inmm²/total intact pre-injury receptive field in mm²²The average percent (and SEM) of the former region of areflexia thatrecovered following PEG treatment at 1 month.³Number of animals recovered/the total number of animals⁴The unit area in pixels comprising the early arriving SSEP peak (seemethods)⁵Comparison of pre and post-injury CAP; Mann Whitney, two tailed test⁶Fisher's exact test, two tailed⁷Mann Whitney, two tailed test

A decrease in the amplitude and extended duration of the CAP is typicalof recovering nerve impulses. Thus, it is both useful and possible tocompare the change in CAP shape before the injury and after recovery todetermine a relative index of the degree of CAP recovery. In this study,the area under the early arriving peak (P1) was measured in pixels inonly PEG-treated animals (since there were no recoveries of SSEPs inControl animals). If 100% of all single nerve fibers contributing to theCAP were once again recruited into conduction subsequent to theinjury—but with a decreased amplitude and extended latency period—thenormalized mean area under the curve (CAP above baseline) divided by thesame pre-injury data should approach unity (1.0). In this experiment,integration of the magnitude (in mVs) and latency (in ms) of PEG-treatedanimal's SSEP P1 divided by the same pre-injury data equaled 0.88(Preinjury Mean=1706, SEM=2583 pixels. Post-PEG mean=11482, SEM=1445pixels, N=10). Paired statistical comparison of these data alsoconfirmed there was not a statistically significant difference in theirmeans, further suggesting limited change in the CAP following PEGmediated recovery (P=0.14, Students T test, paired two tailedcomparison). Altogether these calculations suggest a significantrecruitment of injured nerve fibers into CAP conduction following PEGtreatment that would not have occurred otherwise.

Recovery of the CTM Reflex

The proportion of recovered and unrecovered animals, as well as the unitarea of the recovered CTM receptive fields between controls andPEG-injected animals was quantitatively compared. The unit area of backskin that did not respond to CTM stimulation following the injury—butbefore PEG treatment—was statistically similar in both groups (P=0.81;Mann Whitney, two tailed test; Table 5). Thus, the spinal injuryproduced a similar level of CTM behavioral loss in all animals. In the10 PEG-treated animals, 3 recovered CTM function within 24 hours of theinjection, 3 more within the first week of the treatment, and 7 by twoweeks. The area of recovering backskin of these ten animals continued toincrease in size to week four when the experiment was ended. The meanarea of recovered CTM receptive fields was approximately 33%. Not onecontrol animal of 10 showed spontaneous recovery of any portion of theCTM receptive field during the 1-month of observation (which was firstobserved at week 4). The differenced in the frequency of recoverybetween PEG-injected and sham-injected animals was statisticallysignificant (P≦0.03, Fishers Exact Test, two tailed). Similar resultswere also achieved in a smaller number of spinal animals in response toa single intraperitoneal injection of PEG (data not shown).

Discussion

PEG is well known to be able to fuse numerous single cells in vitro intoone giant cell, as well as join the membranes of neurons and giantinvertebrate axons [Bittner, G. D., Ballinger, M. L., and Raymond, M. A.(1986): Reconnection of severed nerve axons with polyethylene glycol,Brain Research, 367, 351-355; Davidson, R. L. and Gerald, P. S. (1976):Improved techniques for the induction of mammalian cell hybridization bypolyethylene glycol, Somat. Cell Genet., 2, 165-176; OLague, P. H. andHuntter, S. L. (1980): Physiological and morphological studies of ratphechromocytoma calls (PC12) chemically fused and grown in culture,Proc. Nat. Acad. Sci. USA, 77, 1701-1705]. As a “proof of concept” ofthe reparative capability of PEG application, variable amounts ofcompletely severed guinea pig white matter axons were physiologicallyand anatomically reconnected in isolated spinal cord [Shi, R., Borgens,R. B., and Blight, A. R. (1999): Functional reconnection of severedmammalian spinal cord axons with polyethylene glycol, J. Neurotrauma,16, 727-738]. This result is less relevant to clinical spinal cordinjury since transections are rare—but set the stage for further testingof the usefulness of the polymer in severely crushed CNS and PNS nervefiber tracts.

In previous reports it has been shown that the reversal of conductionloss in injured spinal cord was associated with a PEG-mediated sealingof breaches in the nerve membrane produced by mechanical damage [Shi, Rand Borgens, R. B. (2001): Anatomical repair of nerve membranes incrushed mammalian spinal cord with polyethylene glycol, J Neurocytol, inpress]. Breaches in nerve membrane allow the unregulated exchange ofions between the extracellular and intracellular compartments. Thiscauses an immediate local collapse in membrane potential and the failureof nerve impulse conduction through this region of the axon. Thisinitial conduction block accounts for the immediate functional lossfollowing SCI, which becomes permanent due to progressive anatomicaldegeneration of injured nerve fibers and spinal parenchyma—so called“secondary injury” [Young, W. (1993): Secondary injury mechanisms inacute spinal cord injury, J. Emerg. Med., 11, 13-22; Tator, C. H. andFehlings, M. G. (1991): Review of the secondary injury theory of acutespinal cord trauma with emphasis on vascular mechanisms, J. Neurosurg75, 15-26]. The remarkable increases in cytosolic Na+ and Ca++movingdown their concentration gradients into the cell (or local region of itsprocess) through compromised membrane is implicated in the destructionof the cell's cytoskeleton and triggers a cascade of degenerativechanges that unchecked, leads to axotomy, sometimes cell death [Borgens,R. B. (1988): Voltage gradients and ionic currents in injured andregenerating axons, Advances in Neurology, Vol. 47: Functional Recoveryin Neurological Diseases, (Waxman, S. G., ed.), pp. 51-66 Raven Press,New York; Maxwell, W. L. and Graham, D. I. (1997): Loss of axonalmicrotubules and neurofilaments after stretch-injury to guinea pig opticnerve fibers, J Neurotrauma, 14, 603-614]. There is clear evidence thatPEG treatment intervenes in this process by sealing the membrane,quickly restoring its ability to propagate nerve impulses and inhibitingthe progressive dissolution of cells of the spinal cord predicated onthe breakdown of the membrane's barrier properties. This result wasshown using a dye exclusion test where PEG treatment largely inhibitedthe uptake of a horseradish peroxidase (HRP; about 40,000 Daltons)marker into damaged axons of crushed guinea pig spinal cord. This effectwas also independent of axon caliber [Shi, R. and Borgens, R. B. (2001):Anatomical repair of nerve membranes in crushed mammalian spinal cordwith polyethylene glycol, J Neurocytol in press]. This seal produced byPEG is not perfect however, in spite of the recovery of membraneexcitability. Reports have been made that local application of the fastpotassium channel blocker 4-Amionopyridine nearly doubles the magnitudeof the recovered CAP in vitro testing [Shi, R. and Borgens, R. (1999):Acute repair of crushed guinea pig spinal cord by polyethylene glycol,J. Neurophysiology, 81, 2406-2414] suggesting that the PEG-sealed regionof membrane is still leaky to potassium.

Membrane breaches secondary to mechanical damage large enough to permitthe uptake of large molecular weight intracellular labels such ashorseradish peroxidase (HRP)— a common means to introduce such markersinto neurons [Borgens, R. B., Blight, A. R. and Murphy, D. J. (1986):Axonal regeneration in spinal cord injury: A Perspective and newtechnique, J. Comp. Neurol., 250, 157 -167; Malgrem, L. and Olsson,(1977): A sensitive histochemical method for light and electronmicroscope demonstration of horseradish peroxidase, Y. J. Histochem.Cytochem., 25, 1280-1283]-likely progress on to such a size as to leadto secondary axotomy. The destruction of the white matter has beenimplicated as producing a robust signal for the inflammatory processeswhich further destroy the cells and tissues of the spinalcord—essentially collateral damage to healthy cells. The histology ofPEG-treated spinal cord lesions has been compared to controls bycomputer managed quantitative 3 D spinal cord reconstruction techniques[Duerstock, B. S., Bajaj, C. L., Pascucci, V., Schikore, D., Lin, K-N.,and Borgens, R. B. (2000): Advances in three-dimensional reconstructionsof the experimental spinal cord injury, Computer Medical Imaging andGraphics, 24 (6), 389-406]. In these studies a topical application ofPEG produced 1-month-old spinal cord lesions of smaller volume, andpossessing less cavitation than measured in control animals (to bereported elsewhere). These data strongly suggests that polymeric sealingof nerve cell membranes is also reflected in an overall reduction inspinal cord pathology which can be observed many weeks later.

Evaluation of the ability of this agent and other water-soluble membranesealing polymers such as the poloxamers and poloxamines continues[Padanlam, J. T., Bischof, J. C., Cravalho, E. G., Tompkins, R. G.,Yarmush, M. L. and Toner, M. (1994): Effectiveness of Poloxamer 188 inarresting calcein leakage from thermally damaged isolated skeletalmuscle cells. Ann N.Y. Acad. Sci. 92, 111-123; Palmer, J. S., Cromie, W.J. and Lee, R. C. (1998): Surfactant administration reduces testicularischemia-reprefusion injury, J. Urology, 159, 2136-2139; Lee, R., River,L. P., Pan, F. S., Wollmann, L. Jr. and R. L. (1992): Surfactant-inducedsealing of electropermeabilized skeletal muscle membranes in vivo, Proc.Natl. Acad. Sci. U.S.A., 89, 4524-4528] as novel treatments for severeCNS and PNS injury, as well as head injury and stroke.

Since the PEG injection can be made many hours after injury, clinicaltesting of an intravenous (IV) PEG administration to severe, acute,natural cases of paraplegia in dogs has begun [Borgens, R. B., Toombs,J. P., Blight A. R., McGinnis M. E., Bauer, M. S., Widmer, W. R. andCook Jr., W. R. (1993): Effects of applied electric fields on clinicalcases of complete paraplegia in dogs, J. Restorative Neurology andNeurosci., 5, 305-322; Borgens, R. B., Toombs, J. P., Breur, G., Widmer,W. R., Water, D., Harbath, A. M., March, P. and Adams, L. G. (1999): Animposed oscillating electrical field improves the recovery of functionin neurologically complete paraplegic dogs, J. of Neurotrama, 16,639-657]. This means of clinical development is unique to this spinalresearch center and has been previously used to develop two otherlaboratory animal derived treatments for spinal injury [Borgens, R. B.,Toombs, J. P., Breur, G., Widmer, W. R., Water, D., Harbath, A. M.,March, P. and Adams, L. G. (1999): An imposed oscillating electricalfield improves the recovery of function in neurologically completeparaplegic dogs, J. of Neurotrama, 16, 639-657; Blight, A. R., Toombs,J. P., Bauer, M. S. and Widmer, W. R. (1991): The effects of4-aminopyridine on neurological deficits in chronic cases of traumaticspinal cord injury in dogs: a phase I clinical trial, J. Neurotrauma, 8,103-119] into human clinical testing. In this new trial, PEGadministration is an adjunct to the routine management of neurologicallycomplete spinal injured dogs since the polymer can be safely introducedin the IV fluids administered soon after their admission to thehospital. Though this clinical trial is not yet completed, preliminaryobservations are encouraging, and appear to show unexpected recoveriesof varied functions within hours to a few days after PEG injections.

EXAMPLE 9 Behavioral Recovery from Spinal Cord Injury Following DelayedApplication of Polyethylene Glycol

In this example, the behavioral character of the recovered CTM reflexproduced by a delayed application of PEG is evaluated, and confirmsobservations of the physiological recovery of conduction in 100% ofthese spinal injured animals.

Methods and Materials

Surgery and Anesthesia

A total of 29 adult (300 gm) guinea pigs were used in this experiment.They were divided into two groups, PEG treated=15, Sham treated=14. Oneanimal died following surgery in the control group. Guinea pigs wereanesthetized with an intramuscular injection of 100 mg/kg ketamine HCL,and 20 mg/kg xylazine, prior to surgical exposure of the cord by dorsallaminectomy and removeal of the Dura (Borgens et al., 1986, 1990). Astandardized injury was produced using a constant displacement 15 secondcompression of the cord using a specially constructed forceps possessinga detente (Blight, 1991, see also Moriarty et al., 1998). This lesioningprocedure had been calibrated to produce total loss of compound actionpotential (CAP) conduction through the spinal cord injury and behavioralfunctioning of the CTM reflex (Borgens and Shi, 2000). To sedate animalsfor behavioral and physiological testing, guinea pigs were injected with0.1 cc Na⁺ Pentobarbital, 50 mg/ml. All surgical procedures and testingwere carried out under protocols approved by the Purdue UniversityAnimal Care and Use Committee, in accordance with Federal, State, andUniversity guidelines governing animal use in research.

PEG Application

An aqueous solution of PEG (1800 daltons, 50% by weight in distilledwater) was applied with a pateur pipette to the exposed injury for twominutes and removed by aspiration. The region was immediately washedwith isotonic Krebs' solution (NaCl 124 mM, KCL 2 mM, KH₂PO₄ 1.24 mM,MgSO₄ 1.3 mM, CaCL₂ 1.2 mM, dextrose 10 mM, NaHCO₃ 26 mM, sodiumascorbate 10 mM) which was also aspirated to remove excess PEG. Insham—treated animals, the injury site was re-exposed surgically at about7 hours, a control application of water applied, for 2 minutes, followedby a lavage with Krebs' solution, which was removed by aspiration. Thewounds were closed, and animals kept warm with heat lamps until awaking.Guinea pigs were housed individually and fed ad libidum.

Behavioral Analysis

The CTM behavior is observed as a rippling of the animal's backskinfollowing light tactile stimulation. These contractions can be measuredby tattooing a matrix of dots on the animal's shaved back. When the skincontracts towards the point of tactile stimulation—the dots move in thisdirection. The skin movement is dependent on sensory afferentsprojecting as a long tract of axons in each ventral funiculus of thespinal cord (just lateral to the spinothalamic tract) to a nuclei of CTMmotor neurons located at the cervical/thoracic junction [Blight, A. R.,Mcginnis, M. E., and Borgens, R. B. (1990): Cutaneus trunci musclereflex of the guinea pig, J. Comp. Neurol., 296, 614-633; Thierault, E.and Diamond, J. (1998): Noceptive cutaneous stimuli evoke localizedcontractions in a skeletal muscle, J. Neurophys., 60 446-447] (see FIG.12A). The reflex is bilaterally organized into segmentally arrangedreceptive fields, displays little supraspinal control, and is lostfollowing spinal injury producing a region of areflexia below the levelof the lesion [Borgens, R. B., Blight, A. R., and McGinnis, M. E.(1990): Functional recovery after spinal cord hemisection in guineapigs: The effects of applied electric fields, J. Comp. Neurol., 296:634-653; Blight, A. R., Mcginnis, M. E., and Borgens, R. B. (1990):Cutaneus trunci muscle reflex of the guinea pig, J. Comp. Neurol., 296,614-633; Thierault, E. and Diamond, J. (1998): Noceptive cutaneousstimuli evoke localized contractions in a skeletal muscle, J.Neurophys., 60 446-447] (FIG. 12A). Recovery of the CTM reflex withinthis region of areflexia is usually not observed for the life of theanimal following transection, and infrequently (<20%) following severecrush lesions to guinea pig spinal cord [Blight, A. R., Mcginnis, M. E.,and Borgens, R. B. (1990): Cutaneus trunci muscle reflex of the guineapig, J. Comp. Neurol., 296, 614-633; Borgens, R. B. and Shi, R.(2000):Immediate recovery from spinal cord injury through molecular repair ofnerve membranes with polyethylene glycol, FASEB, 14: 27-35].

To visualize and quantify the CTM behavior, we evaluated four individualcomponents of it by stopframe analysis of videotaped records of theperiods of behavioral testing. 1.) the unit area of recovery ofreceptive fields below the level of the lesion, 2) the direction of skinmovement in normally functioning and recovered receptive fieldsfollowing injury, 3.) the distance of skin movement during peakcontraction, and 4) the velocity of skin contraction following tactilestimulation.

The overall pattern of skin movement can be quite complex in response toa very focal stimulus. Thus we chose to restrict our quantitativeevaluation of backskin movement to the peak contractions in response tostimulation at any time point. When the region of peak skin contractionwas determined as the region of skin where dots were displaced thegreatest distance, the video tape was reversed to a time just prior tostimulation and skin movement. Then the video tape was advanced atintervals of {fraction (1/24)}^(th) of a second so that a timepointprior to—and just at the peak of skin contraction—could be captured tothe computer. These frames were superimposed over images of the animals,and the distance of peak contraction divided by the time required toproduce it. This provided a measure of the velocity of skin contraction(FIGS. 24A-24C). The character of skin movement following tactilestimulation was determined at the preinjury evaluation for all but fouranimals, and for all animals at 1 day, 3 days, 2 weeks, and 1 month posttreatment. When the peak contraction was determined for any one animal,a protractor was used to measure the angle in which skin pulled towardsthe monofilament probe used to stimulate the backskin relative to animaginary line perpendicular to the animals long axis at the midline.The peak contraction of skin was recorded as a positive angle whenpulling towards the probe, and a negative angle in the infrequent casewhere skin pulled away from the probe. The peak contraction was alsorecorded to be due to stimulation ipsilateral or contralateral ofmidline relative to the place of tactile stimulation.

Physiological Recording of SSEPs

Subdermal electrodes stimulated nerve impulses from the tibial nerve ofthe hind leg (stimuli trains in sets of 200 at 3 Hz; stimulus amplitude<3 mA square wave, 200 μs in duration; FIGS. 25A-25C). EvokedPotentials, more properly Somatosensory Evoked Potentials (SSEPs), wereconducted through the spinal cord to the sensory cortex of the brain.Recording of SSEPs employed a pair of subdermal electrodes located abovethe level of the contralateral cortex with a reference electrode usuallylocated in the ipsilateral pinna of the ear. The stimulation andcomputer management of evoked potential recordings utilized a NihonKohden Neuropak 4 stimulator/recorder and PowerMac G3 computer. In allanimals, the failure to record an SSEP was further confirmed to be dueto a lack of evoked potential conduction through the lesion by a controltest carried out at this time. The medial nerve of the forelimb wasstimulated, initiating evoked potentials in a neural circuit above thelevel of the crush injury. To perform this test, recording electrodeswere left in place while stimulating electrodes were relocated tostimulate the median nerve of the foreleg.

Computer Management of Behavioral Data

Video images were acquired to an Intel® Dual Pentium® Pro computer.Superimposing of images, the coloring of receptive field boundaries madeon the backskin of the animals during CTM testing, and the generalmanagement of video images was performed using Adobe® Photoshop®software. Final Plates were constructed with Microsoft® PowerPointsoftware and printed on an Epson Stylus Color 800 printer. Quantitativeplanimetry of the unit area of receptive fields—or regions of behavioralloss and recovery from these video images—was carried out using IP LabSpectrum™ software.

Statistics

The Mann Whitney, two tailed test was used to compare the means of thedata derived from experimental and sham-treated groups. To compare theproportions between groups, Fishers exact test was used. All tests wereperformed using INSTAT software.

Results

Only 1 animal died (subsequent to surgery) during the coarse of thisstudy. The loss of the receptive fields subsequent to lesioning of thespinal cord resulted in a region of areflexia that was similar in allanimals (mean loss of the total receptive field in sham treatedanimals=59.2%±5.0; in PEG treated animals=52.2%±2.4. P=0.19; MannWhitney two tailed test).

A second surgery permitted the application of PEG about 7 hours postinjury. A sham treatment (2 minute lavage of the lesion with waterfollowed by aspiration, and a Kreb's lavage) was made to control animalsat this same time. Animals were tested for behavioral and physiologicalrecovery about 24 hours, 3 days, 2 weeks, and 1 month post treatment. Inevery case where an SSEP was not recorded following stimulation of thetibial nerve of the hind leg, the median nerve control procedure wasused to test if this was due to any problem that would result in a“false negative” electrical recording. The control procedure confirmedthe failure of evoked potentials to propagate through the lesion inevery test.

As will be discussed below, since not one control animal recovered anSSEP, 13 of the 15 experimental animals with the best electrical recordswere quantitatively evaluated. Similarly, a full evaluation of CTMfunctioning by stop-frame videographic analysis was carried out on thethree controls that recovered the reflex, and on 13 of the 15 recoveredPEG-treated animals for comparison.

The Cutaneus Trunchi Muscle Reflex

Application of PEG produced a very rapid recovery of CTM function in 73%of treated animals within the first 24 hours compared to a complete lackof spontaneous recovery in sham-treated animals at this time (FIGS. 26Aand 26B). Some spontaneous recovery of the CTM reflex in controls beganto appear on day 3 resulting in 3 recoveries out of a total of 13animals (23%) by one month (Table 6). In marked contrast, 11 of 15PEG-treated animals recovered the reflex activity within the region ofareflexia during the first day post treatment (Table 6, FIGS. 26A and26B), and another three animals by 1 month (total recovery=93%;P<0.0003; Fisher's exact test, two tailed; FIGS. 26A, 26B, 27A, 27B).The unit area of recovered areflexic backskin was 27.6%±8.6 in the 15PEG-treated animals, and 18.3%±3.4 for the three controls. Thus, thetotal area of PEG mediated recovery was not statistically different thanthat which occurs spontaneously—but infrequently (P=0.28; Mann Whitney,two tailed test). TABLE 6 Functional and physiological recovery inexperimental (Exp) and control (Ctl) animals. Cutaneous Trunci MuscleRecovery SSEP Recovery N¹ Post² Day 1³ Day 3 Wk 2 Wk4 Stat⁴ Post² Day 1Day 3 Wk 2 Wk4 Stat³ Exp 15 0 11 12 12 14 P ≦ 0 13 14 14 15 P ≦ Ctl 13 00 1 2 3 .0003 0 0 0 0 0 .0001¹N = total number of animals evaluated²The total number of animals showing CTM functioning when measurementswere made within 30 minutes of surgical lesioning of the spinal cord—butprior to the PEG or sham treatment. Note that both CTM reflexes and SSEPconduction was eliminated in all animals.³The number of animals showing recovery of either CTM functioning orSSEP conduction at the times specified.⁴The P values for all comparisons between control and experimentalanimals at each time point; Fishers Exact Test, two tailed.

In general, back skin contracts towards the point of stimulus when theCTM reflex is activated. The largest response usually occurs ipsilateralto the point of stimulation, as the reflex is largely laid outbilaterally. However, there is a minor contralateral contraction inresponse to ipsilateral stimulation of the skin, thus details of theregion of peak skin contraction on both sides of the midline areprovided with reference to the focal area of stimulation in Table 7.Only 6 examples of 52 separate comparisons (Experimental and Control;left and right side) were observed where the peak contraction wasdirected away from the focal stimulus in the uninjured animal,emphasizing that this is normally an infrequent occurrence. When thedirection of skin contraction, its angle, and velocity were comparedbetween the preinjury and postinjury data in PEG-treated animals, therewas no statistical difference—thus the recovered reflex was faithfullyreproduced by PEG treatment (Table 6).

An evaluation was also made of the change in direction of skincontraction on both left and right sides of the animals by pairedcomparison of the angle of skin contraction in only PEG-treated animals(as will be emphasized below, control CTM behavior did not change at allfollowing its spontaneous recovery and in only three animals). The meanangle of skin contraction following ipsilateral CTM stimulation was notsignificantly different after PEG mediated recovery than the normal CTMin the same animals prior to injury (P=0.43, Mann Whitney, two tailedpaired comparison) as was the contralateral responses (P=0.44, sametest). In the three spontaneous recoveries in control animals, the peakdistance of contraction and its velocity (1 mm, and 25 nm/secrespectively) was identical in two, and the recovered reflex unchanged.In the third, only a reduction of the velocity of CTM contraction wasmeasured while the angle of contraction and the peak distance ofcontraction remained unchanged (Table 6). TABLE 7 Characteristics of CTMrecovery prior to, and 1 month after Injury Cutaneous Trunci MusclePerformance Velocity Range N¹ Direction² Dist³ (mm/sec)⁴ (mm/sec)⁵ ExpPre⁶ 11 10/11; (88.7 ± 1.2) 1.2 ± 0.12 15.7 ± 1.9 8.4-25 Post 15 13/15;(80.7 ± 6.5   1.3 ± 0.17 19.9 ± 4.4 8.4-50 Statistic 0.2 0.62 0.4 — CtlPre 3 3/3 (90°)⁸ 1.0⁸ 25⁸ — Post 3 3/3 (90°)⁸ 1.0 20.8 ± 4.2 — CTM Angleof Contraction Ipilateral Contralateral N X¹⁰ SEM¹⁰ Range Statistic X¹⁰SEM¹⁰ Range Statistic Pre 11 45 18.7 −90/90 P = 0.44 49.4 25.9 −90/900.43 Post 11 70 24.2 −80/90 19.4 25.1 −90/90¹N = total animals in presurgery and postsurgery data sets²The direction and angle of orientation of CTM skin contraction. Thenumber of observations where skin pulled towards the stimulus is givenover the total animals evaluated. The angle of skin contraction isexpressed relative to an imaginary line perpendicular to the long axisof the animal, and the mean angle of contraction and its standard errorfor those animals is given in parenthesis.³The mean distance of peak contraction and its standard error⁴The mean velocity of CTM contraction and its standard error⁵The minimum and maximum velocities for each group are given⁶Four PEG treated animals did not receive a presurgery evaluation.⁷Statistical evaluation, comparing the mean data in each column, usedthe Mann Whitney two tailed test.⁸All three control animals were identical in these measurements, thusthere was no standard error to report.⁹The angle of skin contraction relative to an imaginary lineperpendicular to the long axis of the animal. Contractions pullingtowards the probe were assigned positive numbers, and negative numbersfor contractions pulling away from it. The data is given for animalswhere the peak contraction of backskin was detected on the same side ofthe animal's midline as the stimulus (ipsilateral) and# when the peak contraction was measured to be on the other side of themidline (contralateral). Note that this data is presented for PEGtreated animals only since all three control recoveries were identicaland all peak responses were ipsilateral to the point of stimulation.Note as well that the data obtained prior to surgery (Pre) was notstatistically different from that obtained 1 month post surgery (Post).Recovery of Conduction through the Injured Spinal Cord

As in previous studies, SSEPs usually segregated into 2 peaks followingtibial stimulation in the uninjured animal: an early arriving peak(about 20-35 ms), and a late arriving peak (40-50 ms). Table 6 shows theproportion of animals recovering an SSEP in the experimental population,which was 87% during the first day following PEG treatment, and by week4 had reached 100%. Not one sham-treated animal recovered conductionduring the same period of time (Table 6). FIG. 28A shows a typicalexample of SSEP records for sham treated animals, as well as a mediannerve control procedure. Such control procedures were undertaken for anymeasurement that failed to demonstrate a repeatable SSEP, and in everycase demonstrated that the lack of evoked potentials was due to afailure to conduct them through the lesion. In FIG. 28B, a typicalprocess of PEG mediated recovery is shown. Note that the latency of therecovering evoked potentials was greater than normal in the early stagesof recovery, but gradually declined with time (depicted for the earlyarriving peaks). FIG. 29 shows that normal latency was not reachedduring the 1 month of observation. FIG. 29 also plots the magnitude ofthe early arriving evoked potentials—which recover to more than 50% oftheir pre-injury values.

Discussion

This example confirms that PEG treatment can reverse behavioral loss andconduction loss secondary to severe spinal cord injury within hours.Here the focus has been exclusively on the delayed application of PEG toa standardized spinal cord injury. The constant displacement injurytechnique produces measurable pathology that is not statisticallysignificantly different between individual animals in the group[Moriarty, L. J., Duerstock, B. S., Bajaj, C. L., Lin. K and Borgens, R.B. (1998): Two and three dimensional computer graphic evaluation of thesubacute spinal cord injury, J. Neurologic. Sci., 155: 121-137]. HoweverPEG mediated reversals are so rapid, with functional recoveriesoccurring in sometimes less than an hour, that injured animals can alsoserve as their own controls [Borgens, R. B. and Shi, R.(2000): Immediaterecovery from spinal cord injury through molecular repair of nervemembranes with polyethylene glycol, FASEB, 14: 27-35].

In this example, the character of the behavioral recovery is evaluatedby comparing its characteristics to the pre-injury reflex. The resultsshow that the recovered reflex activity is statistically similar in thedirection, distance, and velocity of CTM contractions when compared tothe normal reflex. The entire receptive field lost after injury was notrestored however. The largest unit area recovered by PEG treatmentapproached 50% of the original area of areflexia. Only three controlanimals showed spontaneous recovery of the CTM reflex. Curiously, theseanimals displayed a recovered CTM that was mostly identical to thepreinjury CTM reflex. This could be due to a slightly less severe injuryin these animals—requiring possibly less robust spontaneous sealing torestore the reflex behavior without any measurable change in it. InPEG-treated animals, there were numerous small alterations in thecharacter of the restored reflex in the treated population—however thesechanges were not statistically significant relative to the preinjury CTMreflex.

In summary, direct application of this hydrophilic polymer to the siteof a spinal cord injury can rapidly reverse behavioral loss restoring anappropriately organized behavior as well as nerve impulse conductionthrough the lesion within a clinically useful time frame.

PEG-Mediated Repair of Neural Injury

This example is one of a series detailed herein that has explored theability of a cell fusogen or biomembrane fusion agent such as PEG toreconnect severed mammalian spinal cord axons, as well as seal theaxolelmma of severely compressed/crushed spinal axons[Shi, R., Borgens,R. B. and Blight, A. R. (1999): Functional reconnection of severedmammalian spinal cord axons with polyethylene glycol, J. Neurotrauma,16: 727-738; Shi, R. and Borgens, R. B. (1999): Acute repair of crushedguinea pig spinal cord by polyethylene glycol, J. Neurophysiology, 81:2406-2414]. The potential mechanisms of action of biomembrane fusionsagents such as PEG are discussed elsewhere herein, as well as thepossibly shared mechanisms with non-ionic triblock polymers such as thepoloxamines and poloxamers discussed below [see Borgens, R. B. and Shi,R.(2000) Immediate recovery from spinal cord injury through molecularrepair of nerve membranes with polyethylene glycol, FASEB, 14: 27-35;Shi, R. and Borgens, R. B. (1999): Acute repair of crushed guinea pigspinal cord by polyethylene glycol, J. Neurophysiology, 81: 2406-2414;Shi, R., Borgens, R. B. and Blight, A. R. (1999): Functionalreconnection of severed mammalian spinal cord axons with polyethyleneglycol, J. Neurotrauma, 16: 727-738]. Briefly; sealing of membranebreaches by high molecular weight molecules such as PEG may involve adehydration of the plasmalemma where closely apposed regions of thebilayer resolve into each other—structural components of the plasmalemmano longer partitioned by the polar forces associated with the aqueousphase. Subsequent to the removal of the fusogen and rehydration, the nowcontinuous phase undergoes spontaneous reassembly. We have shown thatthe restored membrane is repaired sufficient to exclude uptake of alarge molecular weight intracellular tracer [Borgens, R. B. and Shi,R.(2000) Immediate recovery from spinal cord injury through molecularrepair of nerve membranes with polyethylene glycol, FASEB, 14:27-35]—but is still porous to a limited exchange of ions, in particularpotassium [Shi, R. and Borgens, R. B. (1999): Acute repair of crushedguinea pig spinal cord by polyethylene glycol, J. Neurophysiology, 81:2406-2414]. The magnitude of recovered CAPs in spinal cord strips inisolation can nearly be doubled by the addition of the fast potassiumchannel blocker 4 aminopyridine [Shi, R. and Borgens, R. B. (1999):Acute repair of crushed guinea pig spinal cord by polyethylene glycol,J. Neurophysiology, 81: 2406-2414]. Membrane fusion processes are stillan active area of research —particularly as models for endogenousmembrane and vessicle fusion [see Lee, J. and Lentz, B. R. (1997):Evolution of lipid structures during model membrane fusion and therelation of this process to cell membrane fusion, Biochemistry, 36:6251-625; Lentz, B. R. (1994): Induced membrane fusion; potentialmechanism and relation to cell fusion events, Chem. Physiol. Lipids, 73:91-106].

Physical reconnection of axons contained within severed strips of guineapig spinal cord ventral white matter was demonstrated in vitro using adouble sucrose gap isolation and recording chamber. Reconnection ofwhite matter was documented by the immediate recovery of CAPs traversingthe original plane of transection following fusion as well as by thediffusion of two intracellular fluorescent labels and by high-resolutionlight microscopy [Shi, R., Borgens, R. B. and Blight, A. R. (1999):Functional reconnection of severed mammalian spinal cord axons withpolyethylene glycol, J. Neurotrauma, 16: 727-738]. Immediate recovery ofconduction across severe crush lesions to ventral white matter in vitrowas documented by similar techniques [Shi, R. and Borgens, R. B. (1999):Acute repair of crushed guinea pig spinal cord by polyethylene glycol,J. Neurophysiology, 81: 2406-2414]. In all studies, an application of anaqueous solution of PEG (50% by weight in distilled water) was used for2 minutes. No difference in response using PEG solutions prepared withpolymers of 400 to about 3000 MW (ibid.) has been detected, but theviscosity of the solution may still be more important to PEG mediatedrepair than the MW of the polymer in this regard. This may be moreimportant to eventual clinical use if topical applications of PEG to thedamaged neural tissue are required.

Membrane Repair in Other Types of Injury

Non-ionic detergents, so called triblock polymers, are similar to PEG,and are believed to share some mechanisms of action in reversing cellpermeabilization. Their structure usually incorporates a high molecularweight central hydrophobic core, with hydrophilic PEG side chains.Poloxamer 188 has proven to reverse muscle cell death subsequent to highvoltage insult [Lee, R., River, L. P., Pan, F. S., Wollmann, L., (1992)Surfactant-induced sealing of electropermeabilized skeletal musclemembranes in vivo, Proc. Natl. Acad. Sci. U.S.A. 89, 4524-4528].Isolated rat skeletal muscle cells were labeled with an intracellularfluorescent dye which leaked out of the cells after high voltage trauma.This insult was sufficient to disrupt muscle membranes allowing theleakage of the marker in 100% of the control preparations. Treatment ofskeletal muscle cells in vitro with P188 reduced—even eliminated—dyeleakage following the injury. Further in vivo tests extended theseresults since an intravenous injection of P188 produced a physiologicaland anatomical recovery of the rat muscle following electric shock [Lee,R., River, L. P., Pan, F. S., Wollmann, L., (1992) Surfactant-inducedsealing of electropermeabilized skeletal muscle membranes in vivo, Proc.Natl. Acad. Sci. U.S.A. 89, 4524-4528]. This approach has also beentested to reverse cell death in a testicular reperfusion injury model inrats [Palmer, J. S. Cromie, W. L. and Lee, R. C. (1998): Surfactantadministration reduces testicular ischemia-reprefusion injury, J.Urology, 159: 2136-2139] P188 can also seal heat shocked muscle cells invitro. This was shown by an inhibition of calcein dye leakage from cellsinduced by elevated temperature [Padanlam, J. T., Bischof, J. C.Cravalho, E. G. Tompkins, R. G. Yarmush, M. L. and Toner, M. (1994)Effectiveness of Poloxamer 188 in arresting calcein leakage fromthermally damaged isolated skeletal muscle cells, Ann N.Y. Acad. Sci. 92111-123]. P188 also rescues fibroblasts from lethal heat shock[Merchant, F. A., Holmes, H. A., Capelli-Schellpfeffer, M., Lee R. C.and Toner, M. (1988) Poloxamer 188 enhances functional recovery oflethally heat-shocked fibroblasts, J. Surgical Research 74 131-140].Another biocompatible detergent (Poloxamer 1107; administered IV) wasused in an in vivo testicular ischemia—reperfusion injury model in rats,as well inhibiting the leakage of hemoglobin from irradiatederythrocytes [Palmer, J. S. Cromie, W. L. and Lee, R. C. (1998):Surfactant administration reduces testicular ischemia-reprefusioninjury, J. Urology, 159: 2136-2139; Hannig, J., Yu, J., Beckett, M.,Weichselbaum R., and Lee, R. C. (1999): Poloxamine 1107 sealing ofradiopermeabilized erythrocyte membranes, Int. J. Radiat. Biol., 75:379-385]. These studies demonstrate that nonionic biocompatibledetergents and large hydrophilic molecules can reverse permeabilizationof cell membranes, and that they can also be administered through thevascular system to reach damaged target cells.

EXAMPLE 10 Intravenous Hydrophilic Polymer Induces Rapid Recovery fromClinical Paraplegia in Dogs

This example demonstrates a swift, striking, and statisticallysignificant recovery of multiple functions in clinical cases of severe,acute, naturally occurring paraplegia in dogs. Recovery of functionoccurred in response to a combination of topically applied, andintravenously administered, polyethylene glycol (PEG). Recoveries ofsensory and motor functions occurred rapidly and at all time pointsstudied between 3 days and 6-8 weeks post-injury.

Admission and Treatment

Dogs with spinal cord injuries were admitted to the emergency servicesof the University Veterinary Teaching Hospitals (UVTH) at Texas A&MUniversity, College Station, Tex., and at Purdue University, WestLafayette, Ind. An identical protocol for admission, neurologicalevaluation, treatment, and follow up (R. B. Borgens et al., J.Restorative Neurology and Neurosci. 5, 305 (1993); R. B. Borgens et al.,J. Neurotrauma 16, 639 (1999)) was adhered to by each Research Center.In special circumstances, computerized x-ray tomography (CT) imaging wasavailable in addition to routine radiography and myelography at theTexas Center, while electrophysiological study of nerve impulseconduction through the spinal cord lesion by evoked potential testingwas performed at Purdue University.

Each dog received a radiological examination (FIGS. 30A-30D), and athorough, videotaped, neurological examination (FIG. 31A) thatincluded: 1) tests for deep pain in hind limbs and digits, 2)superficial pain appreciation below the level of the injury in flank,lower limbs and digits, 3) proprioceptive evaluation of the hind limbs(i.e. conscious proprioception), 4) evaluation of hind limb load-bearingand voluntary locomotion, and 5) spinal reflex testing (patellar,tibialis, cranialis, flexor withdrawal, and sciatic reflexes). Tests 1-4were also used as functional measures of outcome and were quantitativelyscored using previously reported techniques and methods (R. B. Borgenset al., J. Restorative Neurology and Neurosci. 5, 305 (1993); R. B.Borgens et al., J. Neurotrauma 16, 639 (1999)). These data then provideda total neurological score (TNS) (R. B. Borgens et al., J. RestorativeNeurology and Neurosci. 5, 305 (1993); R. B. Borgens et al., J.Neurotrauma 16, 639 (1999)) for each dog at each time point tested.Since neurological recovery is varied in its expression between animals,the most valid means to compare outcomes is by comparison of the TNS(R.B. Borgens et al., J. Restorative Neurology and Neurosci. 5, 305(1993)). All dogs admitted to the clinical trial possessed the worstclinical signs for spinal injury secondary to spinal cord compressioncharacterized by complete paraplegia, urinary and fecal incontinence,and lack of deep pain response [grade 5 lesions (J. R. Coates, CommonNeurological Problems 30, 77 (2000))]. These functional tests (andothers, see below) were also used as exclusion criterion so thatneurologically “incomplete” dogs were not included in the trial.Additionally, only paraplegic dogs with upper motor neuron syndrome—truespinal cord injuries—were study candidates. A persistent lack, orhyporeflexia of the lower limb(s) revealed segmental compromise ofspinal cord circuitry or “lower motor neuron sequela”. This wassufficient to exclude animals from the study (R. B. Borgens et al., J.Restorative Neurology and Neurosci. 5, 305 (1993); R. B. Borgens et al.,J. Neurotrauma 16, 639 (1999)). During the initial clinical evaluation,owners were asked to review a document concerning the experimentaltreatment, and then requested to sign an informed consent should theywish to participate in the study.

Next, paraplegic dogs received the first of two intravenous injectionsof PEG. Later, but within 24 hours of admission, the location of thelesion was determined by survey radiography and myelography (FIGS.30A-30D). The latter examination insured that myelomalacia was limitedto less than 1 vertebral segment. All dogs received an injection ofmethylprednisilone sodium succinate (30 mg/kg body weight), underwentgeneral anesthesia, and taken to surgery. All injured dogs receivedstandard—of—care veterinary management of these injuries, includingsurgical decompression of the affected site and fixation of thevertebral column when required. The dura was removed duringdecompressive surgery, exposing the spinal cord lesion, and about 1 ccof the PEG solution (about 2000 daltons, 50% W/W in sterile saline; 150mg/kg body weight) was layered onto the injury site. The polymer wasaspirated from the surface of the exposed cord within 2 min ofapplication, next the region lavaged with sterile Ringer's solution, andthese fluids aspirated as well. A fat pad graft was placedsuperficially, the incision closed, and the animals taken to theIntensive Care Unit (ICU) for recovery. Within 24 hours of surgery, asecond injection of PEG, identical to the first, was performed usuallyin ICU. Animals were monitored within the hospital for 7-10 days, and afull neurological exam, videotaped as was the original, was performedapproximately 3 days (74±9 hours) post surgery, approximately 1 weekpost surgery at discharge (6.8 days±1.2 days) and at 6-8 weeks postsurgery at recheck. As in past clinical trials, owners were provideddetailed instructions concerning care of their animals (i.e. bladderexpression, skin care, etc.) and, initially, use of a wheeled cart (K-9Carts, Montana) to aid in the dog's rehabilitation (R. B. Borgens etal., J. Restorative Neurology and Neurosci. 5, 305 (1993); R. B. Borgenset al., J. Neurotrauma 16, 639 (1999)). However this latter practice wasdiscontinued after only 3 admissions because the recovery of functionwas so rapid (see below) such as to make the use of the cartunnecessary.

Control Dogs

During the development of the experimental protocol, paraplegic dogsrecovered rapidly and unexpectedly within a few days after PEGadministration. Participating neurosurgeons believed it unethical tocarry out a control procedure (intravenous injection of the solvent forPEG—sterile saline) knowing full well these client-owned animals wouldsustain variable, but severe, life long behavioral losses (R. B. Borgenset al., J. Neurotrauma 16, 639 (1999)). Given the ca. 48-hour window intreatment, it was also not possible to perform a single cross-overstudy. Thus a medical decision was made to use historical controlsrather than inject such severely injured animals with sterile saltwater. Relevant historical control data was obtained for sham-treateddogs from recent peer reviewed and published studies performed at theIndiana Center (R. B. Borgens et al., J. Restorative Neurology andNeurosci. 5, 305 (1993); R. B. Borgens et al., J. Neurotrauma 16, 639(1999)). These control dogs were 1) also admitted to veterinary clinicaltrials restricted to only neurologically complete cases of acute canineparaplegia, 2) received identical conventional management as describedabove, 3) were neurologically evaluated by identical methods, andexcluded from the studies by identical exclusion criteria (R. B. Borgenset al., J. Restorative Neurology and Neurosci. 5, 305 (1993); R. B.Borgens et al., J. Neurotrauma 16, 639 (1999)) (see FIGS. 30A-30E andFIGS. 32A and 32B) were evaluated at the same time points, and 5) inmost cases, their neurological scores were derived by the sameinvestigators participating in this trial (R. W., G. B., J. T., R. B).It is important to emphasize that all investigators were completelyblinded to the experimental or control status of all dogs recruited intothese previous trials. The use of these identical procedures inrecruitment and particularly in the scoring of neurological functionsyielded little to no variation between the multiple investigators whentheir individual scores were compared (R. B. Borgens et al., J.Restorative Neurology and Neurosci. 5, 305 (1993); R. B. Borgens et al.,J. Neurotrauma 16, 639 (1999)). The validity of this comparison appearsto be eminently greater than data gathered from the veterinaryliterature. These latter investigations do not: i) use multipleneurological functions as exclusion criteria to limit the possibilitythat evaluations would include neurologically incomplete dogs, ii)report a complete axis of neurological behavior including the functionof relevant lower spinal reflexes, iii) use the outcome measures usedhere, or iv) evaluate animals at the same post-surgical time points postsurgery.

For our comparison complete medical records, score sheets, and videotapes were available for 14 control (sham-treated) dogs from 1993 (R. B.Borgens et al., J. Restorative Neurology and Neurosci. 5, 305 (1993))and 11 control dogs from 1999 (3)-25 dogs total for comparison to 20PEG-treated dogs. Moreover, in the latter clinical trial (R. B. Borgenset al., J. Neurotrauma 16, 639 (1999)), the experimental application(oscilating field stimulation) was delayed in 12 experimental dogs forabout 96 hours after surgery to determine what, if any, early functionalrecovery could be associated with surgery and steroid treatment alone.The neurological status of this subset of dogs was reported (R. B.Borgens et al., J. Neurotrauma 16, 639 (1999)). These data then,provided a total of 37 control dogs to compare to 20 PEG-treated dogs atthe 3 day time point, and 25 control dogs for comparison at the 1 weekand 6-8 week neurological checkups.

Clinical Responses to Polymer Administration in Paraplegic Dogs

The most sensitive indicator of early functional recovery in clinicalcases of neurologically complete canine paraplegia is the reappearanceof deep pain response in hind limbs and digits (R. B. Borgens et al., J.Restorative Neurology and Neurosci. 5, 305 (1993); R. B. Borgens et al.,J. Neurotrauma 16, 639 (1999); J. R. Coates, Common NeurologicalProblems 30, 77 (2000)). This was evaluated in 17 of the 20 acutelyinjured PEG-treated dogs approximately 3 days after surgery(approximately 48 hours after the second injection of PEG (FIG. 31A).During this time, 4 of the 17 PEG-treated dogs recovered deep pain,while only one of the 37 control dogs had (P=0.03; Fisher's exact test,two tailed, in this and all subsequent comparison of proportions).

Comparison of the mean TNS at this time, a numbers derived largely fromrecoveries in deep and superficial pain, was markedly statisticallysignificantly improved in the PEG-treated group compared to controls(P=0.009; comparison of means here and below were made using a Students'T test, two tailed, or the Welch variation, FIGS. 30A-30E).

Though more than half of the population of PEG-treated dogs hadrecovered deep pain responses by 1 week post-treatment, improvements indeep pain in 25 control dogs eliminated significance in this onefunctional comparison between the groups at this time point (P=0.2).However, recoveries in proprioception, improvement in load bearing inhind limbs and voluntary walking in 8 PEG-treated dogs of 20 at thistime were unmatched by such improvements in control dogs. Analysisrevealed marked statistically significant improvement in the TNSs ofPEG-treated dogs at this time point (P=0.007; FIGS. 30A-30E).

The total neurological scores of control dogs showed modest andprogressive improvement by the 6-week recheck, however, this remainedmanifest as mainly improvements in the quality of pain appreciation (R.B. Borgens et al., J. Restorative Neurology and Neurosci. 5, 305 (1993);R. B. Borgens et al., J. Neurotrauna 16, 639 (1999)). Thus, there was nosignificant difference between the PEG-treated and control dogs when theproportions of animals with positive deep and superficial pain responseswere compared (P=0.1 and 0.6, respectively). However, this improvementin pain appreciation was not matched by any of the other outcomemeasures evaluated in control dogs. Thirty-five percent (7 of 20) of thePEG-treated dogs recovered measurable proprioception by 6 weeks, whileonly 1 of 25 (4%) of control animals had improved proprioception(P=0.01). Fully 70% of all PEG-treated dogs (14 of 20) could ambulatevoluntarily, compared to only 28% (7 of 25) of controls (P=0.007).Furthermore, the overall quality of functional recovery secondary to PEGadministration at the 6 week recheck (as given by the total neurologicalscore) was strikingly improved by PEG treatment relative to controls(P≦0.0008; FIGS. 30A-30E).

Qualitatively, the two groups appeared quite different in a mannermasked by the dry recitation of quantitative neurological scores andproportions. The possible range of an individual dog's totalneurological score was 4 (a totally paraplegic dog) to 20 (a totallynormal dog (see FIGS. 30A-30E). Fifteen of the 25 control dogs (60 %)remained neurogically complete paraplegics 6-8 weeks after decompressivesurgery and corticosteroid treatment, all were individually assessed aneurological score of 4. The best performing control dog scored 11 atthis time point (R. B. Borgens et al., J. Neurotrauma 16, 639 (1999)).However, this animal remained seriously impaired; locomotion alone wasevaluated as only a score of 2. PEG treatment resulted in 35% (7 dogs ofthe 20) individually scoring 13 to 16. By the 6-8 week recheck some dogshad made a such a striking recovery—to the extent any remainingfunctional loss could only be determined by a thorough neurologicalexamination. Only 3 of 20 PEG-treated dogs (15%) remained paraplegic atthe end of the 6-8 week period of observation (a highly significantcomparison to controls, P=0.003).

Electrophysiology and Bladder Management

Evoked potential testing [Somatosensory Evoked Potential or SSEP (R. B.Borgens et al., J. Restorative Neurology and Neurosci. 5, 305 (1993); R.B. Borgens et al., J. Neurotrauma 16, 639 (1999)), FIGS. 31A-31C] wasperformed on II of the 12 PEG-treated dogs recruited to the PurdueCenter to determine if nerve conduction through the lesion had beenrestored (FIGS. 31A-31C). Somatosensory Evoked Potential recordingscould not usually be obtained prior to the first PEG injection andsurgery due to the need to move these animals through the battery ofevaluations and on to surgery as soon as possible after admission. Inaddition, most dogs were unable to be sedated for such tests in thefirst few hours after admission due to food intake and othercomplicating factors. Of the 11 dogs on which electrophysiological testsof conductance were performed at more than two recheck periods, 7 wererecorded to have positive SSEPs, while 4 did not demonstrate evidence ofnerve impulse conductance through the lesion. All four dogs scoringabove the median TNS of 12 showed a clear recovery of conductancethrough the lesion. Furthermore, one severely injured animal(fracture/dislocation and subluxation of the vertebral column) wasaccessible for SSEP testing during the second of two PEG injections.This animal showed a progression from a negative SSEP (flatline) to lowamplitude, long duration, cortical potentials during the 30 min periodof injection and observation of the sedated animal (FIGS. 32A, 32B).

In contrast, of the 11 control dogs from the 1993 study, none recoveredSSEP conduction by even 6 months post injury [refer to page 313 (R. B.Borgens et al., J. Restorative Neurology aid Neurosci. 5, 305 (1993))].Only two control dogs of 14 recovered measurable conduction by 6-8months in the 1999 clinical trial [refer to page 649 (R. B. Borgens etal., J. Neurotrauma 16, 639 (1999))]. This proportion of PEG-treateddogs (7 of 11) that recovered ascending electrophysiological conductionthrough the lesion was highly statistically significant compared to therelative lack of recorded evoked potentials in control dogs (P=0.001).

The status of bladder continence due to paraplegia is problematic indogs just as in man. We have found that electrophysiologicalmeasurements of micturation (urethral pressure profilemetry andcystometry) while providing data relevant to isolating cases of lowermotor neuron syndrome, do not correlate highly with observations ofrecovery of urinary continence particularly by owners (R. B. Borgens etal., J. Neurotrauma 16, 639 (1999)). Incontinence is not easily confusedby owners, since it represents a major behavioral loss in the dog's“house training” and is the most common reason given for euthanasia oftheir pets. Moreover, a consistent failure of owners to manually expressthe bladder of incontinent dogs leads to readmission for urinary tractinfection. Of the 20 PEG-treated dogs, owners reported all but 6 werecontinent, and did not require bladder expression. These latter animalswere of a group of PEG treated dogs exhibiting the least recovery at theend of the study. We offer these facts as additional but modest evidencethat recovery from paraplegia mediated by PEG likely improved oreliminated at least urinary incontinence as well.

Paraplegia in Laboratory Animals, Dogs, and Man

The history of spinal cord injury research can be characterized in somepart by the quest for standardized injury methods and credible means toassay behavioral loss and recovery in laboratory animals—usually adultguinea pigs, rats, or cats. There has always been debate and controversyconcerning both of these quests. In the former, the difficulty centerson various different techniques used to induce injury to the exposedspinal cord. For example, constant impact [usually standardized weightdrop techniques (S. K. Somerson, and B. T. Stokes, Exp. Neurol. 96, 82(1987))], constant compression of the spinal cord [using speciallyfabricated clips (A. S. Rivln, C. H. Tator, Surg. Neurol. 10, 39 (1978))or forceps (A. R. Blight. J. Neurologic. Sci. 103, 156 (1990))] andpartial or complete transection (R. B. Borgens, A. R. Blight, D. J.Murphy, J. Comp. Neurol. 250, 157 (1986)), of the spinal cord have beenemployed and contrasted (R. B. Borgens, A. R. Blight, D. J. Murphy, J.Comp. Neurol. 250, 157 (1986)). With the exception of the lattertechnique, an important goal of these methods has been to reduce thevariability in lesions, and to produce a central hemorrhagic injurytypical of clinical injury in man (A. R. Blight. J. Neurologic. Sci.103, 156 (1990); A. R. Allen, J. Am. Med. Assoc. 57, 878 (1911); C. H.Tator, M. J. Fehlings, Neurosurg. 75, 15, (1991)). While the successesof the different approaches can be debated relative to these goals,there is no question that modern laboratory injuries are made to thesurgically exposed spinal cords of anesthetized animals producing aninitially dorsal locus of injury. This is inconsistent with mostclinical injuries where the initial site of SCI (“spinal cord injury”)injury is anterior (ventral), and the impact is to the trunk of the bodyor neck (so called “closed” injuries). Moreover, during experimentalinsult to the cord, anesthesia provides neuroprotection (S. K. Salzman,M. M. Mendez, S. Sabato, et al., Brain Res. 521, 33 (1990)), and is acomplicating factor. Thus, naturally-occurring injuries in dogs providea more direct comparison to clinical injuries in man (R. B. Borgens, inSpinal Cord Dysfunction, Volume III: Functional Stimulation, L. S.Illis, Ed. (Oxford Medical Publications, Oxford, 1992), chap 5).

There have also been numerous and varied attempts to measure and/orquantitate the behavioral recovery from SCI in laboratory models ofspinal injury. Measurement of hindlimb locomotion (M. D. Basso, M.Beattie, J. D. Bresnahan, J. Neurotrauma 12, 1 (1995)), or some form ofit (A. S. Rivilin, C. H. Tator, J. of Neurosurgery 47, 577-581 (1977)),has dominated rodent studies of SCI—usually because of the underlyingnotion that the results might be relevant to lower limb locomotion inman even though there is no evidence to support such a view. Humans arethe only obligatory bipedal mammals, and upright walking is completelydominated by supraspinal control (S. Mori, K. Matsuyama, E. Miyashita,K. Nakajima, M. Asanome, Folia Primatologica 66, 192 (1996)). Inexperimental SCI models, locomotion is dominated by locally controlledand generated stepping (S. Rossignol, R. Dubuc, Curr. Opin. Neurobiol.4, 894-902 (1994); A. Naito, Y. Shimuza, Y. Handa, Neurosci Res 8, 281(1990)). Such walking behavior is often called “spinal walking” toseparate it from walking behavior that requires the restoredtransmission of nerve impulses through the spinal cord lesion fromhigher centers. Because restored nerve impulse traffic through thelesion is not required for voluntary ambulation in animals, walkingbehavior by itself does not represent a valid behavioral recovery withwhich to infer restored conduction through the lesion. This requires useof kinestheseological methods confirming fore limb and hind limbcoordination during voluntary locomotion.

For all of the above reasons, no attempt has been made to develop evenmore complicated systems to grade walking behavior associated withclinical paraplegia in dogs. Instead, reliance is placed on a simple 5point score that provides a reliable, precise reflection of increasingcapabilities in ambulation, but without additionally attempting toindicate the neural mechanisms of action underlying it (R. B. Borgens etal., J. Restorative Neurology and Neurosci. 5, 305 (1993); R. B. Borgenset al., J. Neurotrauma 16, 639 (1999)).

In the present example, whatever the mechanism underlying the return ofvoluntary walking—a strikingly significant number of dogs walked withsuperior capability than occurred in controls. Moreover, thestatistically significant improvement in TNS is a clear indication ofsubstantive, and meaningful recovery in several other clinicallyrelevant areas of function, including: recovery in the neurologicalappreciation of both deep and superficial pain, recovery of ascendingnerve impulse conductance through the lesion, recovery of consciousproprioception, as well as substantial load bearing and voluntarywalking.

The strengths of these methods as applied to naturally occuringparaplegia are that they provide real potential for assessing theclinical importance of experimental therapies for human SCI (R. B.Borgens et al., J. Neurotrauma 16, 639 (1999); A. R Blight, J. P.Toombs, M S. Bauer, W. R. Widmer, J. Neurotrauma 8, 103-119 (1991)). Theweakness of this SCI model is that little is learned about thebiological basis for the response to treatment. This is more easilyachieved in laboratory models where invasive physiological testing andanatomical techniques can be applied (R. B. Borgens, in Spinial CordDysfunction, Volume III: Functional Stimulation, L. S. Illis, Ed.(Oxford Medical Publications, Oxford, 1992), chap 5).

Polymer Application in Experimental SCI

Both topical and/or intravascular administration of polyethylene glycol(2000-3000 Daltons, approximately 30-50% WIW in water) has beendocumented to induce:

-   -   1) Rapid (minutes) anatomical fusion of severed white matter        axons (R. Shi, R. B. Borgens, A. R. Blight, J. Neurotraum 16,        727 (1999)) and rapid sealing of anatomic breaches in both        myelinated and unmyelinated axons of guinea pig ventral white        matter (R. Shi, R. B. Borgens, J. Neurophysiology 81, 2406        (1999)). In both cases neural tissue was maintained and        evaluated ill vitro in a double sucrose gap recording chambers        (R. Shi, A. R. Blight, Neuroscieince 77, 553-562 (1997)).    -   2) Rapid (minutes) recovery of nerve impulse conduction through        the lesion in these same studies (R. Shi, R. B. Borgens, A. R.        Blight, J. Neurotraun 16, 727 (1999); R. Shi, R. B. Borgens, J.        Neurophysiology 81, 2406 (1999))- or through severe and        standardized crush injuries to the guinea pig spinal cord in        vivo, measured by SSEP testing (R. B. Borgens, R. Shi, FASEB 14,        27 (2000); R. B. Borgens, D. M. Bohnert, J. Neurosci. Res. 66,        1179 (2001); R. B. Borgens, R. Shi, D. M. Bohnert, J. Exp. Bio.        205, 1(2002)).    -   3) Rapid (hours to days) recovery of long-tract dependent spinal        cord reflex (the cutaneous trunchi muscle or CTM reflex) (R. B.        Borgens, R. Shi, FASEB 14, 27 (2000); R. B. Borgens, D. M.        Bohnert, J. Neurosci. Res. 66, 1179 (2001); R. B. Borgens, R.        Shi, D. M. Bohnert, J. Exp. Bio. 205, 1 (2002)), which is        totally dependent on the integrity of an identified white matter        column of axons within the ventral funiculus of the guinea pig        (A. R. Blight, M. E. McGinnis, R. B. Borgens, J. Comp. Neurol.        296, 614-633 (1990)) and rat (E. Thierault, J. Diamond, J.        Neurophysiol. 60, 446-447 (1988)) spinal cord.

A variable level of recovery of the CTM reflex (produced by compressionof the spinal cord) occurred in>90% of PEG-treated guinea pigs, comparedto a range of 0-17% in sham-treated control populations in threeseparate studies (R. B. Borgens, R. Shi, FASEB 14, 27 (2000); R. B.Borgens, D. M. Bohnert, J. Neurosci. Res. 66, 1179 (2001); R. B.Borgens, R. Shi, D. M. Bohnert, J. Exp. Bio. 205, 1 (2002)). Therecovery of cortical potentials was documented as restored volleys ofSSEPs measured to arrive at the sensory motor cortex followingelectrical stimulation of the tibial nerve of the hind limb. In all(100%) of the control guinea pigs, such nerve impulse conduction throughthe lesion was eliminated for the 1 month of observation. In PEG-treatedanimals, SSEPs recovered in 100 % of the population in these same threeinvestigations (R. B. Borgens, R. Shi, FASEB 14, 27 (2000); R. B.Borgens, D. M. Bohnert, J. Neurosci. Res. 66, 1179 (2001); R. B.Borgens, R. Shi, D. M. Bohnert, J. Exp. Bio. 205, 1 (2002)).

Mechanisms of Polymer Based Therapy for Neurological Injuries

The molecular mechanisms of action of, for instance, surfactants andtri-block polymers in sealing or fusing cell membranes have beenreviewed in the literature. (R. B. Borgens, Neurosurgery 49, 370-379(2001); B. R. Lentz, Chem. Phys. Lipid 73, 91 (1994); J. Lee, B. R.Lentz, Biochemistry 36, 6251 (1997); J. M. Marks, C—Y. Pan, T. Bushell,W. Cromie, R. C. Lee FASEB J 15, 1107 (2001).) Briefly: an initialmechanism common to all hydrophilic surfactants that may be beneficialto soft tissue trauma is the formation of a chemical film sealingdefects in the cell membranes at the site of mechanical damage. However,it is the watery-hungry character of this class of hydrophilic polymers(PEG, EPAN, and some dextrans) that is believed to instantly dehydratethe membrane locally. Furthermore, either removal or rearrangement ofwater molecules in the vicinity of membrane breach permits the lipidcore of the intact membrane surrounding the breach—and perhaps thestructural elements suspended in it—to merge into each other. When thepolymer is removed, or in lowered concentration, variable amounts ofstructural self-assembly occur in response to reintroduction of theaqueous phase of the membrane. Triblock polymers such as poloxamers arecomprised largely of PEG—yet they also possess a hydrophobic component(polypropylene oxide) which may actually target breaches inmembranes—inserting into the breach where the hydrophobic core of themembrane is exposed (J. M. Marks, C—Y. Pan, T. Bushell, W. Cromie, R. C.Lee FASEB J 15, 1107 (2001)). The long PEG side chains likely contributeto sealing in the fashion described above. We have tested poloxamer 188in a spinal injury model in guinea pigs and have found no difference inthe physiological and behavioral recoveries in response to PEG asdescribed above. These findings suggest various polymers may provebeneficial for application to soft tissue trauma and other injuries tothe nervous system (J. M. Marks, C-Y. Pan, T. Bushell, W. Cromie, R. C.Lee FASEB J 15, 1107 (2001); J. Donaldson, R. Shi, R. Borgens,Neurosurgery 50, 147-157 (2002)).

Likely any large molecular polymer like PEG or poloxamers, introduced tothe blood supply, will target only regions of tissue trauma where thereis a loss of vascular integrity. We have demonstrated this by observingaccumulation of a flourescently labeled PEG in crushed guinea pig spinalcord—comparing intraveneous, subcutaneous, and peritoneal administrationwith a topical application of the polymer to the exposed lesion (R. B.Borgens, D. M. Bohnert, J. Neurosci. Res. 66, 1179 (2001)). Labeling wasbarely detectable or non-existent in intact regions of the spinal cordin these same animals.

Of the putative mechanisms of action for PEG, formal proof of itsmembrane sealing properties have been demonstrated. The uptake ofextracellular applied labels such as horseradish peroxidase (HRP),ethidium bromide, or the leakage of lactic dehydrogenase into theextracellular space, are excellent indices of cell membrane compromise(R. Shi, R. B. Borgens, J. Neurocytology 29, 633-643 (2000)). Bothuptake of, and leakage of, these intracellular labels from injured whitematter of the spinal cord is strikingly reduced or eliminated by PEGadministration. Furthermore, the susceptibility for axonal sealing isequal across a broad range of axon calibers (R. Shi, R. B. Borgens, J.Neurocytology 29, 633-643 (2000)).

We hypothesized this inhibition of leakage of the nerve fiber membranereduces the opportunity for secondary axotomy to occur. This isconsistent with the observation that PEG-treated cords are more intact,possess greater amounts of intact white matter, and a reduced lesionvolume than in untreated guinea pig spinal cord as shown by quantitativecomparison of three dimensional reconstructions of these spinal cords(B. S. Duerstock, R. B. Borgens, J. Exp. Biol. 205, 13 (2002)).

In summary, intraveneous and topical administration of a hydrophilicpolymer in clinical cases of acute neurologically complete spinal cordinjury in dogs results in an unexpected, rapid recovery of multiplemeasures of functional outcome. Such a rapid and complete clinicalrecovery is not observed in response to conventional clinical/surgicalmanagement of neurologically complete injuries, including theadministration of steroids, and decompressive surgery (J. R. Coats et.al., Veterinary Surgery 24, 128-139 (1995)).

While the invention has been illustrated and described in detail in thedrawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly the preferred embodiment has been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected. In addition, all references cited hereinare indicative of the level of skill in the art and are herebyincorporated by reference in their entirety.

1. A method for treating an injury to nerve tissue of a mammalianpatient, comprising administering an effective amount of a biomembranefusion agent comprising an amphipathic polymer to the patient so thatthe biomembrane fusion agent is delivered via the patient's vascularsystem to the site of the injured nerve tissue.
 2. The method of claim1, wherein said amphipathic polymer comprises a block copolymer.
 3. Themethod of claim 2, wherein said block copolymer is taken from the groupconsisting of polyalkylene glycol block copolymers, mixtures ofpolyalkylene glycol block copolymers, and mixtures of polyalkyleneglycols and polyalkylene glycol block copolymers.
 4. The method of claim2, wherein said block copolymer comprises at least one compound takenfrom the group consisting of polymethylene glycol, polyethylene glycol,polypropylene glycol, polybutylene glycol, polypentylene glycol,polyhexylene glycol, polyheptylene glycol, polyoctylene glycol,polynonylene glycol, and polydecylene glycol, and branched andstructural isomers thereof.
 5. The method of claim 1, wherein saidamphipathic polymer is taken from the group consisting of polyethyleneglycol/polypropylene glycol block copolymers, and mixtures ofpolyethylene glycol, polypropylene glycol, and polyethyleneglycol/polypropylene glycol block copolymers.
 6. The method of claim 1,wherein said amphipathic polymer is taken from the group consisting ofpoloxamers and poloxamines.
 7. The method of claim 1, wherein saidbiomembrane fusion agent is in a pharmaceutically acceptable carrier. 8.The method of claim 1, wherein said carrier is water.
 9. The method ofclaim 1, wherein the administering of said effective amount of saidbiomembrane fusion agent includes injecting said biomembrane fusionagent into a vascular system of the patient.
 10. The method of claim 1,wherein the administering of said effective amount of biomembrane fusionagent includes injecting said biomembrane fusion agent subcutaneouslyinto the patient.
 11. The method of claim 1, wherein the administeringof said effective amount of biomembrane fusion agent includes injectingsaid biomembrane fusion agent intraperitoneally into the patient. 12.The method of claim 1, wherein the administering of said effectiveamount of biomembrane fusion agent includes injecting said biomembranefusion agent into or near the nerve sheath at site of injury.
 13. Themethod of claim 12, wherein the injured nerve tissue is peripheral nervetissue.
 14. The method of claim 1, wherein the injured nerve tissue isspinal cord tissue.
 15. The method of claim 1, wherein the injured nervetissue is peripheral nerve tissue.
 16. The method of claim of claim 1,wherein the injury comprises an injury selected from the groupconsisting of a mechanical injury, a biochemical injury and an ischemicinjury.
 17. The method of claim 16, wherein the injury comprises anischemic injury.
 18. The method of claim 17 wherein the ischemic injuryis selected from the group consisting of a stroke and a head injury. 19.A method for treating an injury to nerve tissue of a mammalian patient,comprising administering an effective amount of a biomembrane fusionagent comprising a poloxamer or a poloxamine to the patient so that thebiomembrane fusion agent is delivered via the patient's vascular systemto the site of the injured nerve tissue, wherein delivery of saidbiomembrane fusion agent is effected using a technique selected from thegroup consisting of intravascular, intramuscular, subcutaneous andintraperitoneal injection.
 20. The method of claim 19 wherein the injurycomprises an injury selected from the group consisting of a mechanicalinjury, a biochemical injury and an ischeric injury.
 21. A method fortreating an injury of to nerve tissue of a mammalian patient, comprisingadministering an effective amount of a biomembrane fusion agentcomprising an amphipathic polymer topically at the site of nerve tissueinjury.
 22. The method of claim 21, wherein the amphipathic polymercomprises a block copolymer.
 23. The method of claim 22, wherein saidblock copolymer is taken from the group consisting of polyalkyleneglycol block copolymers, mixtures of polyalkylene glycol blockcopolymers, and mixtures of polyalkylene glycols and polyalkylene glycolblock copolymers.
 24. The method of claim 22, wherein said blockcopolymer comprises at least one compound taken from the groupconsisting of polymethylene glycol, polyethylene glycol, polypropyleneglycol, polybutylene glycol, polypentylene glycol, polyhexylene glycol,polyheptylene glycol, polyoctylene glycol, polynonylene glycol, andpolydecylene glycol, and branched and structural isomers thereof. 25.The method of claim 21, wherein said amphipathic polymer is taken fromthe group consisting of polyethylene glycol/polypropylene glycol blockcopolymers, and mixtures of polyethylene glycol, polypropylene glycol,and polyethylene glycol/polypropylene glycol block copolymers.
 26. Themethod of claim 21, wherein said amphipathic polymer is taken from thegroup consisting of poloxamers and poloxamines.
 27. The method of claim21, wherein said biomembrane fusion agent is in a pharmaceuticallyacceptable carrier.
 28. The method of claim 21, wherein said carrier iswater.